Monomeric and dimeric fluorescent protein variants and methods for making same

ABSTRACT

The present invention relates generally to fluorescent proteins and fluorescent protein variants, and more specifically to monomeric and dimeric forms of Anthozoan fluorescent proteins. In one aspect, the present invention provides variants of fluorescent proteins, where the variants have a reduced propensity to tetramerize, and form dimeric or monomeric structures. In a further aspect, the present invention provides variants of fluorescent proteins, the variants being characterized by more efficient maturation than corresponding fluorescent proteins from which they are derived. The invention also relates to methods of making and using such fluorescent proteins and fluorescent protein variants, including fluorescent protein monomers and dimers.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/931,304 filed Aug. 30, 2004, which is a continuation-in-part of U.S.patent application Ser. No. 10/209,208 filed Jul. 29, 2002, which is acontinuation-in-part application of U.S. patent application Ser. No.10/121,258 filed Apr. 10, 2002, which is a continuation-in-part of U.S.patent application Ser. No. 09/866,538, filed May 24, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/794,308,filed Feb. 26, 2001, and claims priority under 35 U.S.C. §120 to all ofwhich applications, the disclosures of which are hereby expresslyincorporated by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made in part with Government support under Grant No.NS27177, awarded by the National Institute of Neurological Disorders andStroke, and under Grant No. GM62114-01, awarded by the NationalInstitute of General Medical Sciences. The Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to variant fluorescent proteins,and more specifically to Anthozoan fluorescent proteins that have areduced propensity to oligomerize, where such proteins form monomericand/or dimeric structures. The invention also relates to methods ofmaking and using such fluorescent protein monomers and dimers. Inparticular, the present invention relates generally to variant redfluorescent proteins (RFPs), and more specifically to Anthozoanfluorescent proteins (AnFP), having at least one amino acid alterationthat results in more efficient maturation than the correspondingwild-type protein or another variant RFP from which such variantsderive. The invention further concerns RFP variants that additionallyhave reduced propensity tetramerize, and thus form predominantlymonomeric and/or dimeric structures. The invention also relates tomethods of making and using such RFP variants.

2. Description of the Related Art

The identification and isolation of fluorescent proteins in variousorganisms, including marine organisms, has provided a valuable tool tomolecular biology. The green fluorescent protein (GFP) of the jellyfishAequorea victoria, for example, has become a commonly used reportermolecule for examining various cellular processes, including theregulation of gene expression, the localization and interactions ofcellular proteins, the pH of intracellular compartments, and theactivities of enzymes.

The usefulness of Aequorea GFP has led to the identification of numerousother fluorescent proteins in an effort to obtain proteins havingdifferent useful fluorescence characteristics. In addition, spectralvariants of Aequorea GFP have been engineered, thus providing proteinsthat are excited or fluoresce at different wavelengths, for differentperiods of time, and under different conditions. The identification andcloning of a red fluorescent protein from Discosoma coral, termed DsRedor drFP583, has raised a great deal of interest due to its ability tofluoresce at red wavelengths.

The DsRed from Discosoma (Matz et al., Nature Biotechnology 17:969-973[1999]) holds great promise for biotechnology and cell biology as aspectrally distinct companion or substitute for the green fluorescentprotein (GFP) from the Aequorea jellyfish (Tsien, Ann. Rev. Biochem.,67:509-544[1998]). GFP and its blue, cyan, and yellow variants havefound widespread use as genetically encoded indicators for tracking geneexpression and protein localization and as donor/acceptor pairs forfluorescence resonance energy transfer (FRET). Extending the spectrum ofavailable colors to red wavelengths would provide a distinct new labelfor multicolor tracking of fusion proteins and together with GFP (or asuitable variant) would provide a new FRET donor/acceptor pair thatshould be superior to the currently preferred cyan/yellow pair (Mizunoet al., Biochemistry 40:2502-2510 [2001]). However, there are problemsassociated with the use of DsRed as a fluorescent reporter, includingits slow and inefficient chromophore maturation and its tendency tooligomerize.

All coelenterate fluorescent proteins cloned to date display some formof quaternary structure, including the weak tendency of Aequorea greenfluorescent protein (GFP) to dimerize, the obligate dimerization ofRenilla GFP, and the obligate tetramerization of the Discosoma DsRed(Baird et al., Proc. Natl. Acad. Sci. USA 97:11984-11989 [2000]; andVrzheshch et al., FEBS Lett., 487:203-208 [2000]). While the weakdimerization of Aequorea GFP has not impeded its acceptance as anindispensable tool of cell biology, the obligate tetramerization ofDsRed has greatly hindered its development from a scientific curiosityto a generally applicable and robust tool, most notably as geneticallyencoded fusion tag. Thus, one problem with DsRed is its tendency tooligomerize.

DsRed tetramerization presents an obstacle—for the researcher who wishesto image the subcellular localization of a red fluorescent chimera, asthe question exists as to what extent will fusing tetrameric DsRed tothe protein of interest affect the location and function of the latter.Furthermore, it can be difficult in some cases to confirm whether aresult is due, for example, to a specific interaction of two proteinsunder investigation, or whether a perceived interaction is an artifactcaused by the oligomerization of fluorescent proteins linked to each ofthe two proteins under investigation. There have been several publishedreports (see, e.g., Mizuno et al., Biochemistry 40:2502-2510 [2001]; andLauf et al., FEBS Lett., 498:11-15 [2001]) and many unpublishedanecdotal communications, in which DsRed chimeras have been described asforming intracellular aggregates that have lost their biologicalactivity. In addition to its tendency to oligomerize, DsRed also suffersfrom slow and incomplete maturation (Baird et al., Proc. Natl. Acad.Sci. USA 97:11984-11989 [2000]).

One approach to overcome these shortcomings has been to continue thesearch for DsRed homologues in sea coral and anemone; an approach thathas yielded several red shifted proteins (Fradkov et al., FEBS Lett.,479:127-130 [2000]; and Lukyanov et al., J Biol. Chem., 275:25879-25882[2000]). However, the fundamental problem of tetramerization has yet tobe overcome. The only published progress towards decreasing theoligomeric state of a red fluorescent protein involved an engineeredDsRed homologue, commercially available as HcRed1 (CLONTECH), which wasconverted to a dimer with a single interface mutation (Gurskaya et al.,FEBS Lett., 507:16-20 [2001]). Although HcRed1 has the additionalbenefit of being 35 nm red-shifted from DsRed, it is limited by a lowextinction coefficient (20,000 M−1 cm⁻¹) and quantum yield (0.015)(CLONTECH Laboratories Inc., (2002) Living Colors User Manual Vol. II:Red Fluorescent Protein [Becton, Dickinson and Company], p. 4) makingthe protein problematic to use in experimental systems.

A methionine at position 66 of a tetrameric nonfluorescent chromoproteinfrom Anemonia sulcata was converted to a fluorescent protein through theintroduction of two mutations (Lukyanov, K. A., Fradkov, A. F.,Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P.; Markelov,M. L., Zaraisky, A. G., Zhao, X., Fang, Y. et al. (2000) J. Biol. Chem.275, 25879-25882). Introduction of a methionine at position 66apparently improved the fluorescent properties of both a green (zFP506)and a cyan (amFP486) tetrameric fluorescent protein though no detailshave been published (Yanushevich, Y. G., Staroverov, D. B., Savitsky, A.P., Fradkov, A. F., Gurskaya, N. G., Bulina, M. E., Lukyanov, K. A. &Lukyanov, S. A. (2002) FEES Lett. 511, 11-14).

Most previous attempts to improve the rate and/or extent of maturationof DsRed (Verkhusha et al., J. Biol. Chem., 276:29621-29624 [2001]; andTerskikh et al., J. Biol. Chem., 277:7633-7636 [2002]) including thecommercially available DsRed2 (CLONTECH, Palo Alto, Calif.), haveprovided only modest improvements. Recently, an engineered. variant ofDsRed, known as T1 has become available, and which matures more rapidly,but appears to suffer from an incomplete maturation (Bevis and Glick,Nat. Biotechnol., 20:83-87 [2002]). Thus; like DsRed, a significantfraction of the T1 protein remains as the green fluorescent intermediatein the aged tetramer.

Thus, there exists a need in the art for the development of redfluorescent polypeptides for use in scientific applications withouttechnical limitations due to oligomerization, especiallytetramerization, and due to inefficient and slow chromophore maturation.There exists a need for methods to produce fluorescent proteins havingreduced propensity for oligomerization, especially reduced propensityfor tetramerization. There exists a need for methods to produce redfluorescent proteins (RFPs) exhibiting more efficient chromophorematuration than wild-type RFPs or other RPF variants. Furthermore, thereexists a need for RFPs that additionally have reduced propensity foroligomerization, such as, e.g., tetramerization. There also exists aneed for RFP variants with improved efficiency of maturation thatdemonstrate useful fluorescence in a monomeric state in experimentalsystems. There exists a need for methods to produce fluorescent proteinsthat demonstrate useful fluorescence in a monomeric state inexperimental systems. The present invention satisfies these needs andprovides additional advantages.

BRIEF SUMMARY OF THE INVENTION

Variants of red fluorescent proteins (RFPs) that have a reducedpropensity to oligomerize are disclosed herein. For example, the variantRFPs disclosed in the present application have a propensity to formmonomers and dimers, where the native form of the RFP has a propensityto form tetrameric structures.

Aspects of the present invention concern variants of red fluorescentproteins (RFPs) comprising at least one amino acid alteration resultingin more efficient chromophore maturation than a corresponding wild-typeor variant RFP. For example, in one aspect, the present inventionconcerns RFP variants that have a reduced propensity to form tetramersas compared to a corresponding wild-type or variant RF. Thus, certainRFP variants of the invention have a propensity to form monomers anddimers, although the native form of the RFP has a propensity to formtetrameric structures. In some embodiments, the present inventionconcerns RFP variants that, in addition to showing more efficientchromophore maturation, also have a reduced propensity to formtetramers. In some aspects, the present invention further concerns RFPvariants that show have improved quantum yield, improved extinctioncoefficient, or improvements in other measurable characteristics of afluorescent protein as compared to a corresponding wild-type or variantRF. Some aspects of the present invention concern RFP variants that showmore efficient maturation, have a reduced propensity to form tetramers,and in addition have improved quantum yield, improved extinctioncoefficient, or improvements in other measurable characteristics of afluorescent protein as compared to a corresponding wild-type or variantRF.

In some aspects, the invention concerns an Anthozoan fluorescent protein(AnFP) having a reduced propensity to oligomerize, comprising at leastone mutation within the wild-type AnFP amino acid sequence that reducesor eliminates the ability of the fluorescent protein to tetramerizeand/or dimerize, as the case may be. The AnFP preferably is the redfluorescent protein of Discosoma (DsRed) of SEQ ID NO: 1, but is by nomeans so limited. In some embodiments, the invention concerns anAnthozoan fluorescent protein (AnFP), e.g., DsRed, comprising at leastone amino acid substitution within the AB and/or AC interface of saidfluorescent protein (e.g., DsRed) that reduces or eliminates the degreeof oligomerization of said fluorescent protein.

In other embodiments, the variant AnFP (e.g., DsRed) having a reducedpropensity to oligomerize is a monomer in which the interfaces betweenthe oligomeric subunits are disrupted by introducing mutations; e.g.,substitutions, which interfere with oligomerization (includingdimerization), and, if necessary, introducing further mutations neededto restore or improve fluorescence which might have been partially orcompletely lost as a result of disrupting the interaction of thesubunits.

Aspects of the invention specifically include dimeric and monomericvariants of other fluorescent proteins in addition to DsRed, such asfluorescent proteins from other species and fluorescent proteins thathave fluorescence emission spectra in wavelengths other than red. Forexample, green fluorescent proteins and fluorescent proteins fromRenilla sp. find equal use with the invention. Furthermore, fluorescentproteins that normally have the propensity to form tetramers and/ordimers find equal use with the invention.

In a particular embodiment, the fluorescent protein is DsRed, and aDsRed variant having a reduced propensity to oligomerize (in this case,tetramerize) is prepared by first replacing at least one key residue inthe AC and/or AB interface of the wild-type protein, thereby creating adimer or monomer form, followed by the introduction of furthermutation(s) to restore or improve red fluorescence properties.

In one aspect, the invention provides variant fluorescent proteins,including but not limited to DsRed, comprising amino acid substitutionsrelative to the respective wild-type sequences, where the substitutionsimpart the advantageous properties to the polypeptide variants. Theseamino acid substitutions can reside at any position within thepolypeptide, and are not particularly limited to any type ofsubstitution (conservative or non-conservative). In one embodiment, themutations restoring or improving fluorescence are amino acidsubstitutions within the plane of the chromophore and/or just above theplane of the chromophore and/or just below the plane of the chromophoreof the fluorescent protein.

In one aspect, the invention provides a polynucleotide sequence encodinga Discosoma red fluorescent protein (DsRed) variant having a reducedpropensity to oligomerize, comprising one or more amino acidsubstitutions at the AB interface, at the AC interface, or at the AB andAC interfaces of the wild-type DsRed amino acid sequence of SEQ ID NO:1, where the substitutions result in reduced propensity of the DsRedvariant to form tetramers, wherein said variant displays detectablefluorescence of at least one red wavelength. In one embodiment, thisprotein sequence has at least about 80% sequence identity with the aminoacid sequence of SEQ ID NO: 1. In another embodiment, the fluorescentprotein has detectable fluorescence that matures at a rate at leastabout 80% as fast as the rate of fluorescence maturation of wild-typeDsRed of SEQ ID NO: 1, while in another embodiment the protein hasimproved fluorescence maturation relative to DsRed of SEQ ID NO: 1. Instill another embodiment, the protein substantially retains thefluorescence properties of DsRed of SEQ ID NO: 1.

In some embodiments, the fluorescent protein variant has a propensity toform dimers. Some proteins contain substitutions in the AB interface andform an AC dimer.

In some embodiments, the fluorescent protein variant comprises aminoacid substitutions that are at one or more of the residues 2, 5, 6, 21,41, 42, 44, 117, 125, and 217 of SEQ ID NO: 1.

In some embodiments, the fluorescent protein variant comprises at leastnine amino acid substitutions that are at residues 2, 5, 6, 21, 41, 42,44, 117, and 217, and additionally at least one more substitutionincluding substitution at residue 125 of SEQ ID NO: 1. The protein canoptionally further comprise at least one additional amino acidsubstitution that is at residue 71, 118, 163, 179, 197, 127, or 131 ofSEQ ID NO: 1. In some embodiments, any one or more of said substitutionsis optionally selected from R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71A, C117T, F118L, I125R, V127T, S131P, K163Q/M, S179T, S197T, andT217A/S.

The invention provides fluorescent protein variants that can be theproteins dimer1, dimer1.02, dimer1.25, dimer1.26, dimer 1.28, dimer1.34,dimer1.56, dimer1.61, or dimer1.76, as provided in FIGS. 20A-D. In someembodiments, the protein variant is dimer2 (SEQ ID NO: 6), or dimer2.2MMM (also termed dimer3 or dTomato) (SEQ ID NO: 81), or tdTomato (SEQID NO: 106).

The invention also provides fluorescent protein variants that can bevariants of the protein dimer2 (SEQ ID NO: 6). A variant of dimer2 (SEQID NO:6) may have about 80% sequence identity, or about 90%, or about95% sequence identity with SEQ ID NO:6, and may comprise one or moreamino acid substitutions selected from amino acid substitutions atpositions 22, 66, 105, and 124, and may also include terminal amino acidadditions or substitutions comprising one or more amino acids homologousto the N- and/or C-terminal amino acids of GFP (e.g., SEQ ID NO: 14 atthe N-terminus and/or SEQ ID NO: 91 or SEQ ID NO:110 at the C-terminus).In still other embodiments, the substitutions in the dimeric protein isoptionally selected from one or more of V22M, Q66M, V104L, and F124M.For example, in an embodiment, the protein variant is dimer2.2MMM(dimer3) (dTomato) (SEQ ID NO: 81).

In some embodiments, the fluorescent dimeric protein variant has atleast about 90% sequence identity with the amino acid sequence of SEQ IDNO: 1, while in other embodiments, the protein has at least about 95%sequence identity with the amino acid sequence of SEQ ID NO: 1.

In still other embodiments, the fluorescent protein variant is amonomer. In this embodiment, the amino acid substitutions are in the ABinterface and the AC interface.

In some embodiments, the monomeric protein variant comprises at least 14amino acid substitutions that are at residues 2, 5, 6, 21, 41, 42, 44,71, 117, 127, 163, 179, 197, and 217, and additionally at least one moresubstitution that is at residue 125 of SEQ ID NO: 1. In otherembodiments, the monomeric protein optionally further comprises at leastone additional amino acid substitution at residue 83, 124, 125, 150,153, 156, 162, 164, 174, 175, 177, 180, 192, 194, 195, 222, 223, 224,and 225 of SEQ ID NO: 1. In still other embodiments, the substitutionsin the monomeric protein is optionally selected from R2A, K5E, N6D,T21S, H41T, N42Q, V44A, V71A, K83L, C117E/T, F124L, I125R, V127T, L150M,R153E, V156A, H162K, K163Q/M, L174D, V175A, F177V, 5179T, I180T, Y192A,Y194K, V195T, S197A/T/I, T217A/S, H222S, L223T, F224G, L225A.

In some embodiments, the monomeric protein variant is selected frommRFP0.1, mRFP0.2, mRFP0.3, mRFP0.4a, mRFP0.4b, mP11, mP17, m1.01, m1.02,mRFP0.5a, m1.12, mRFP0.5b, m1.15, m1.19, mRFP0.6, m124, m131, m141,m163, m173, m187, m193, m200, m205 and m220, as provided in FIGS.20A-20D. In some embodiments, the monomeric variant is mRFP1 (SEQ ID NO:8), or is mRFP1.5 (SEQ ID NO: 83) or is another monomeric variant.

In some embodiments, the monomeric variant is a variant of mRFP1 (SEQ IDNO: 8) or related fluorescent proteins. A variant of mRFP1 (SEQ ID NO:8) may have 80%, or 90%, or 95% sequence identity with SEQ ID NO: 8, andmay comprise one or more amino acid substitutions selected from aminoacid substitutions at positions 7, 17, 21, 32, 66, 77, 78, 83, 108, 125,147, 150, 161, 163, 174, 177, 182, 194, 195, 196, 197, 199 and 213, andmay also include terminal amino acid additions or substitutions selectedfrom one or more amino acids homologous to the amino acids at the GFPterminus (e.g., SEQ ID NO: 14, SEQ ID NO:91 and SEQ ID NO:91), the aminoacids DNMA, and the amino acids NNMA. Such insertions are preferablyafter amino acid E6. In still other embodiments, the substitutions inthe monomeric protein is optionally selected from one or more of V7I,R17H, T21S, E32K, Q66T/M, A77T/P, D78G, L83F/M, T108A, R125H, T147S,M150L, I161V, M163Q, D174S, V177T, M182K, K1941, T195V/A/L, D195A,D196G, I197E/Y, L1991, and Q213L. In some embodiments, the proteinvariant is selected from mRFP1.5 (SEQ ID NO: 83), OrS4-9 (SEQ ID NO:85), Y1.3 (mYOFP1.3) (mBanana) (SEQ ID NO: 87), mFRFP (F2Q6) (mGrape2)(SEQ ID NO: 89), mRFP2 (mCherry) (SEQ ID NO: 92), mOFP (74-11) (SEQ IDNO: 94), mROFP (A2/6-6) (SEQ ID NO: 96), mStrawberry (SEQ ID NO:98),mTangerine (SEQ ID NO: 100), mOrange (mOFP1) (SEQ ID NO: 102), mHoneydew(SEQ ID NO:104), and mGrape1 (SEQ ID NO: 108).

In some embodiments, the monomeric variant has at least about 90%sequence identity with the amino acid sequence of SEQ ID NO: 1, while inother embodiments, the protein has at least about 95% sequence identitywith the amino acid sequence of SEQ ID NO: 1.

The present invention also provides tandem dimer forms of DsRed,comprising two DsRed protein variants operatively linked by a peptidelinker. The peptide linker can be of variable length, where, forexample, the peptide linker is about 10 to about 25 amino acids long, orabout 12 to about 22 amino acids long. In some embodiments, the peptidelinker is selected from GHGTGSTGSGSS (SEQ ID NO: 17), RMGSTSGSTKGQL (SEQID NO: 18), and RMGSTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19).

In some embodiments, the tandem dimer subunit is selected from dimer1,dimer1.02, dimer1.25, dimer1.26, dimer 1.28, dimer1.34, dimer1.56,dimer1.61, dimer1.76, dimer2, dimer 2.2MMM (dimer 3) (dTomato) SEQ IDNO: 81) and tdTomato (SEQ ID NO: 106), as provided in FIGS. 20A-20D andFIGS. 32-33. The tandem dimer can be a homodimer or a heterodimer. Insome embodiments, the tandem dimer comprises at least one copy of dimer2(SEQ ID NO: 6), dimer 2.2MMM (dimer 3) (dTomato) (SEQ ID NO: 81), or maybe tdTomato (SEQ ID NO: 106).

The present application also provides fusion proteins between anyprotein of interest operatively joined to at least one fluorescentprotein variant of the invention. This fusion protein can optionallycontain a peptide tag, and this tag can optionally be a polyhistidinepeptide tag.

The present application also provides polynucleotides that encode eachof the fluorescent protein variants described or taught herein.Furthermore, the present invention provides the fluorescent proteinvariants encoded by any corresponding polynucleotide described or taughtherein. Such polypeptides can include dimeric variants, tandem dimervariants, or monomeric variants.

In other embodiments, the invention provides kits comprising at leastone polynucleotide sequence encoding a fluorescent protein variant ofthe invention. Alternatively, or in addition, the kits can provide thefluorescent protein variant itself.

In other embodiments, the present invention provides vectors that encodethe fluorescent protein variants described or taught herein. Suchvectors can encode dimeric variants, tandem dimer variants, or monomericvariants, or fusion proteins comprising these variants. The inventionalso provides suitable expression vectors. In other embodiments, theinvention provides host cells comprising any of these vectors.

In another embodiment, the invention provides a method for thegeneration of a dimeric or monomeric variant of a fluorescent proteinwhich has propensity to tetramerize or dimerize, comprising the steps ofmutagenizing at least one amino acid residue in the fluorescent proteinto produce a dimeric variant, if the protein had the propensity totetramerize, and a monomeric variant, if the protein had the propensityto dimerize; and mutagenizing at least one additional amino acid residueto yield a dimeric or monomeric variant, which retains the qualitativeability to fluoresce in the same wavelength region as thenon-mutagenized fluorescent protein.

In an optional variation of this method, an additional step can beadded, essentially introducing a further mutation into a dimeric variantproduced from a fluorescent protein that had the propensity to formtetramers to produce a monomeric variant. In some embodiments, thisadditional step can come after the first mutagenizing step.

In some embodiments, this method can result in dimeric or monomericvariants having improved fluorescence intensity or fluorescencematuration relative to the non-mutagenized fluorescent protein.

The mutagenesis used in the present method can be by multiple overlapextension with semidegenerate primers, error-prone PCR, site directedmutagenesis, or by a combination of these. The results of thismutagenesis can produce protein variants that have a propensity to formdimers or monomers.

In some embodiments of this method, the fluorescent protein is anAnthozoan fluorescent protein, and optionally, the Anthozoan fluorescentprotein fluoresces at a red wavelength. The Anthozoan fluorescentprotein can be Discosoma DsRed.

In other embodiments, the fluorescent protein variants of the inventioncan be used in various applications. In one embodiment, the inventionprovides a method for the detection transcriptional activity, where themethod uses a host cell comprising a vector encoding a variant DsRedfluorescent protein operably linked to at least one expression controlsequence, and a means to-assay said variant fluorescent proteinfluorescence. In this method, assaying the fluorescence of the variantfluorescent protein produced by the host cell is indicative oftranscriptional activity.

In other embodiments the invention also provides a a polypeptide probesuitable for use in fluorescence resonance energy transfer (FRET),comprising at least one fluorescent protein variant of the invention.

In still another embodiment, the invention provides a method for theanalysis of in vivo localization or trafficking of a polypeptide ofinterest, where the method uses a fluorescent fusion protein of theinvention in a host cell or tissue, and where the fusion protein can bevisualized in the host cell or tissue.

In a further aspect, the invention concerns further improved variants ofred fluorescent proteins (RFPs) that have reduced propensityto-oligomerize. In particular, the invention concerns RFP variants thatnot only have a propensity to form monomers and dimers, where the nativeform of the RFP has a propensity to form tetrameric structures, but areadditionally characterized by more efficient maturation than thecorresponding non-oligomerizing variants from which they derive. Suchvariants are typically brighter than the corresponding non-oligomerizingvariants, where brightness is typically expressed as the product of theextinction coefficient (EC) and the quantum yield (QY) at the desiredred wavelength.

The invention further provides a polynucleotide encoding a variant of ared fluorescent protein (RFP) having a propensity to form tetramericstructures, comprising at least one amino acid alteration resulting inmore efficient maturation into the desired red species from the immaturegreen species, and at least one further amino acid alteration resultingin a reduced propensity to tetramerize. The amino acid alteration may besubstitution, insertion and/or deletion, and preferably is substitution.

Thus, in one aspect the invention concerns a polynucleotide encoding avariant of a red fluorescent protein (RFP) having a propensity to formtetrameric structures, comprising at least one amino acid alterationresulting in higher fluorescence intensity at red wavelength, and atleast one further amino acid alteration resulting in a reducedpropensity to tetramerize.

In a particular embodiment, the polynucleotide encodes a Discosoma redfluorescent protein (DsRed) variant.

In another embodiment, the polynucleotide encodes a DsRed variant, inwhich the amino acid substitution resulting in more efficient maturationis at position 66 of wild-type DsRed of SEQ ID NO: 1. A particularsubstitution is a Q66M substitution within SEQ ID NO: 1. In anotherembodiment, the polynucleotide encodes a DsRed variant, in which theamino acid substitution resulting in more efficient maturation is atposition 147 of wild-type DsRed of SEQ ID NO: 1. The substitution atposition 147 preferably is a T147S substitution, but other substitutionsare also possible at this position and are specifically included withinthe scope of the present invention. In a further embodiment, thepolynucleotide of the invention encodes a DsRed variant comprising asubstitution at both position 66 and position 147 within SEQ ID NO: 1.In a preferred embodiment, the polynucleotide of the invention encodes aDsRed variant comprising a Q66M and a T147S substitution.

The polynucleotides of the invention encoding DsRed variants comprisingat least one amino acid alteration resulting in improved efficiency ofchromophore maturation into the desired red form, such as, for example,a Q66M substitution, may additionally contain codons for any of theother amino acid substitutions, alone or in any combination, discussedhereinabove and throughout the present disclosure, in connection withother embodiments. In particular, such polynucleotides (e.g. thoseencoding a DsRed variant with a Q66M mutation alone or in combinationwith a T147S substitution) may encode DsRed variants further comprisingone or more substitutions at the AB interface, at the AC interface, orat the AB and AC interfaces of the wild-type DsRed amino acid sequenceof SEQ ID NO: 1, where the substitutions result in reduced propensity ofthe DsRed variant to form tetramers.

In a particular embodiment, such polynucleotides encode DsRed variantsadditionally comprising one or more substitutions at an amino acidposition selected from the group consisting of 42, 44, 71, 83, 124, 150,163, 175, 177, 179, 195, 197, 217, 2, 5, 6, 125, 127, 180, 153, 162,164, 174, 192, 194, 222, 223, 224, 225, 21, 41, 117, and 156 within thewild-type DsRed amino acid sequence of SEQ ID NO: 1. Possiblesubstitutions at the indicated positions include, without limitation,one or more substitutions selected from the group consisting of N42Q,V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T,S1971, T217A, R2A, K5E, N6D, I125R, V127T, I180T, R153E, H162K, A164R,L174D, Y192A, Y194K, H222S, L223T, F224G, L225A, T21S, H41T, C117E, andV156A within the wild-type DsRed amino acid sequence of SEQ ID NO: 1.

Thus, polynucleotides encoding DsRed variants comprising the followingsubstitutions: N42Q, V44A, V71A, K83L, F124L, L150M, K163M, V175A,F177V, S179T, V195T, S1971, T217A, R2A, K5E, N6D, I125R, V127T, I180T,R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G, L225A,T21S, H41T, C117E, and V156A within the wild-type DsRed amino acidsequence of SEQ ID NO: 1, are specifically within the scope of theinvention.

Preferred dimeric embodiments of the invention include a polynucleotideencoding a dimer2 (SEQ ID NO:6), a polynucleotide encoding a dimer2.2MMM(dimer3) (dTomato) (peptide SEQ ID NO: 81, DNA SEQ ID NO: 82), and apolynucleotide encoding a tdTomato (peptide SEQ ID NO: 106, DNA SEQ IDNO: 107). Preferred monomeric embodiments of the invention include apolynucleotide encoding a mRFP1.1 shown in FIG. 30 (SEQ ID NO: 79), apolynucleotide encoding a mRFP1.5 (SEQ ID NO: 83), a polynucleotideencoding a OrS4-9 (SEQ ID NO: 85), a polynucleotide encoding a Y1.3 (SEQID NO: 87), a polynucleotide encoding a F2Q6 (SEQ ID NO: 89), apolynucleotide encoding a mRFP2 (mCherry) (SEQ ID NO: 92), apolynucleotide encoding a mOFP (74-11) (SEQ ID NO: 94), a polynucleotideencoding a mROFP (A2/6-6) (SEQ ID NO: 96), a polynucleotide encoding amStrawberry (SEQ ID NO:98), a polynucleotide encoding a mTangerine (SEQID NO: 100), a polynucleotide encoding a mOrange (mOFP1) (SEQ ID NO:102), a polynucleotide encoding a mHoneydew (SEQ. ID NO:104), and apolynucleotide encoding a mGrape1 (SEQ ID NO:108).

In another aspect, the invention concerns a polynucleotide encoding afusion protein, comprising at least one DsRed protein variant encoded bythe polynucleotides discussed above, operatively joined to at least oneother polypeptide of interest. In particular embodiments, such fusionproteins may comprising either the Q66M or T147S substitution, or both,as a tandem dimer.

The invention further concerns polypeptides encoded by thepolynucleotides discussed above, vectors containing such polynucleotides(including expression vectors), and recombinant host cells transformedwith such polynucleotides or vectors.

The invention, in a different aspect, concerns a kit comprising at leastone polynucleotide or polypeptide discussed above.

In yet another aspect, the invention concerns a method for the detectiontranscriptional activity, comprising:

(a) providing a host cell comprising a vector, wherein said vectorcomprises nucleotide sequence encoding a DsRed fluorescent proteinvariant comprising at least one amino acid alteration resulting inhigher fluorescence intensity at red wavelength, and at least onefurther amino acid alteration resulting in a reduced propensity totetramerize operably linked to at least one expression control sequence,and a means to assay said variant fluorescent protein fluorescence, and

(b) assaying fluorescence of said variant fluorescent protein producedby said host cell, where variant fluorescent protein fluorescence isindicative of transcriptional activity.

In a further aspect, the invention concerns a method for the detectionof protein-protein interactions, comprising detection of energy transferfrom a fluorescent or bioluminescent protein fusion to a fusion proteinas discussed above.

In a still further aspect, the invention concerns a method for theanalysis of in vivo localization or trafficking of a polypeptide ofinterest, comprising the steps of:

(a) providing a polynucleotide encoding a fusion protein, comprising atleast one DsRed protein variant encoded by the polynucleotides discussedabove, operatively joined to at least one other polypeptide of interestand a host cell or tissue, and

(b) visualizing said fusion protein that is expressed in said host cellor tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the tetrameric form of DsRed (PDB identification code1G7K). The A-C and B-D interfaces are equivalent, as are the A-B and C-Dinterfaces.

FIGS. 2A-2C show graphical representations of the tetramer, dimer andmonomer forms of DsRed, respectively, based on the x-ray crystalstructure of DsRed. Residues 1-5 were not observed in the crystalstructure but have been arbitrarily appended for the sake ofcompleteness. The DsRed chromophore is represented in red and the fourchains of the tetramer are labeled following the convention of Yarbroughet al. (Yarbrough et al., Proc. Natl. Acad. Sci. USA 98:462-467 [2001]).FIG. 2A shows the tetramer of DsRed with the residues mutated in T1indicated in blue for external residues and green for those internal tothe β-barrel. FIG. 2B shows the AC dimer of DsRed with all mutationspresent in dimer2 represented as in FIG. 2A and the intersubunit linkerpresent in tdimer2(12) shown as a dotted line. FIG. 2C shows the mRFP1monomer of DsRed with all mutations present in mRFP1 represented as inFIG. 2A.

FIGS. 3A-3C show the results of an analytical ultracentrifugationanalysis of DsRed, dimer2, and mRFP0.5a polypeptides, respectively. Theequilibrium radial absorbance profiles at 20,000 rpm were modeled with atheoretical curve that allowed only the molecular weight to vary. TheDsRed absorbance profile (FIG. 3A) was best fit with an apparentmolecular weight of 120 kDa, consistent with a tetramer. The dimer2absorbance profile (FIG. 3B) was best fit with an apparent molecularweight of 60 kDa, consistent with a dimer. The mRFP0.5a absorbanceprofile (FIG. 3C) was best fit with an apparent molecular weight of 32kDa, consistent with a monomer containing an N-terminal polyhistidineaffinity tag.

FIGS. 4A-4D show fluorescence and absorption spectra of DsRed, T1,dimer2 and tdimer2(12) and mRFP1, respectively. The absorbance spectrumis shown with a solid line, the excitation with a dotted line and theemission with a dashed line.

FIG. 5 shows a maturation time course of red fluorescence for DsRed, T1,dimer2, tdimer2(12) and mRFP1. The profiles are color coded, asindicated in the key. Log phase cultures of E. coli expressing theconstruct of interest were rapidly purified at 4° C. Maturation at 37°C. was monitored beginning at 2 hours post-harvest. The initial decreasein mRFP1 fluorescence is attributed to a slight quenching on warmingfrom 4 to 37° C.

FIGS. 6A-6F show light and fluorescence microscopic images of HeLa cellsexpressing Cx43 fused with T1, dimer2 or mRFP1. Images 6A, 6C and 6Ewere acquired with excitation at 568 nm (55 nm bandwidth) and emissionat 653 nm (95 nm bandwidth) with additional transmitted light. Luciferyellow fluorescence (images 6B, 6D and 6F) was acquired with excitationat 425 nm (45 nm bandpass) and emission at 535 nm (55 nm bandpass). FIG.6A shows two contacting cells transfected with Cx43-mRFP1 and connectedby a single large gap junction. FIG. 6B shows one cell microinjectedwith lucifer yellow at the point indicated by an asterisk and the dyequickly passing (1-2 sec) to the adjacent cell. FIG. 6C shows fourneighboring cells transfected with Cx43-dimer2. The bright line betweenthe two right-most cells is the result of having two fluorescentmembranes in contact and is not a gap junction. FIG. 6D showsmicroinjected dye slowly passing to an adjacent cell (observedapproximately one third of the time). FIG. 6E shows two adjacent cellstransfected with Cx43-T1 and displaying the typical perinuclearlocalized aggregation. FIG. 6F shows no dye passed between neighboringcells.

FIG. 7 shows a schematic representation of the directed evolutionstrategy of the present invention. Randomization at two positions isshown but the technique has been used with up to five fragments.

FIGS. 8A and 8B show SDS-PAGE analysis of DsRed, T1, dimer2,tdimer2(12), and mRFP1 polypeptides. The oligomeric state of eachprotein is demonstrated by running each protein (20 μg) both not boiledand boiled on a 12% SDS-PAGE Tris-HCl precast gel (BioRad). FIG. 8Ashows the gel prior to Coomasie staining, which was imaged withexcitation at 560 nm and emission at 610 nm. The tandem dimertdimer2(12) has a small tetrameric component due a fraction of thecovalent tandem pairs participating in intermolecular dimer pairs.Fluorescent proteins that are not boiled do not necessarily migrate attheir expected molecular weight. FIG. 8B shows the same gel as in FIG.8A after Coomasie staining. The band at −20 kDa results from partialhydrolysis of the mainchain acylimine linkage in protein containing ared chromophore.

FIGS. 9A-9D show fluorescent images of red fluorescent proteinsexpressed in E. coli. E. coli strain JM109(DE3) was transformed witheither DsRed, T1, dimer2 or mRFP1, plated on LB/agar supplemented withampicillin, and incubated 12 hours at 37° C. then 8 hours at 20° C.before the plate was imaged with a digital camera. In FIG. 9A, thequadrants corresponding to T1, dimer2, and mRFP1 all appear of similarbrightness when excited at 540 nm and imaged with a 575 nm (long pass)emission filter. Almost no fluorescence is visible for identicallytreated E. coli transformed with DsRed. In FIG. 9B, when excited at 560nm and imaged with a 610 (long pass) filter, mRFP1 appears brighter dueto its 25 nm red shift. In FIG. 9C, the monomer mRFP1 does not contain agreen fluorescent component and is thus very dim in comparison to T1 anddimer2 when excited at 470 nm, a wavelength suitable for excitation ofEGFP. FIG. 9D shows a digital color photograph of the same plate takenafter 5 days at room temperature reveals the orange and purple hues ofT1 and mRFP1, respectively.

FIGS. 10A and 10B show a table describing the protocols and multiplelibraries created during evolution of dimer1 and mRFP1, as well as otherintermediate forms. The templates, method of mutagenesis, targetedpositions within the DsRed polypeptide, and resulting clones areindicated.

FIGS. 11A-11C show a table providing a key to the primer pairs used inthe mutagenesis protocols, as well as the target codon positions.

FIGS. 12A and 12B provide the PCR primer sequences listed in FIGS.11A-11C.

FIG. 13 shows a table describing the results of a series of experimentstesting the functionality of various DsRed chimeric molecules. Thechimeric molecules comprise a DsRed sequence and the Cx43 polypeptide.The plasmids encoding the fusion polypeptides were transfected into HeLacells, and the ability of the expressed fusion polypeptides to formfunctional gap junctions was assayed by the microinjection of luciferyellow dye. Passage of the dye from one HeLa cell to an adjacent HeLacell indicates the presence of a functional gap junction, and thus, afunctional fusion polypeptide.

FIG. 14 shows various biophysical properties of wild-type DsRed, T1,dimer2, tdimer2(12), and mRFP1 polypeptides.

FIG. 15 shows a table providing excitation/emission wavelength values,relative maturation speed and red/green ratio values of red and greenfluorescent protein species.

FIG. 16 provides the nucleotide sequence of the Discosoma sp. wild-typered fluorescent protein open reading frame (DsRed) (SEQ ID NO:2).

FIG. 17 provides the amino acid sequence of the Discosoma sp. wild-typered fluorescent protein (DsRed) (SEQ ID NO:1).

FIG. 18 provides the nucleotide sequence of the Discosoma sp. variantfast T1 red fluorescent protein (SEQ ID NO:5).

FIG. 19 provides the amino acid sequence of the Discosoma sp. variantfast T1 red fluorescent protein (SEQ ID NO:4).

FIGS. 20A-20D provide a table showing the amino acid substitutionsidentified during the construction of variant DsRed proteins. Also shownare the substitutions originally contained in the fast T1 DsRed variant.

FIG. 21 provides the nucleotide sequence of the Discosoma variant redfluorescent protein dimer2 open reading frame (SEQ ID NO:7).

FIG. 22A provides the amino acid sequence of the Discosoma variant redfluorescent protein dimer2 (SEQ ID NO:6).

FIG. 22B provides the amino acid sequence of the GFP termini andillustrates their locations at the N-terminal (SEQ ID NO: 14) andC-terminal (SEQ ID NO: 91) ends of a fluorescent protein.

FIG. 23 provides the nucleotide sequence of the Discosoma variant redfluorescent protein mRFP1 open reading frame (SEQ ID NO:9).

FIG. 24 provides the amino acid sequence of the Discosoma variant redfluorescent protein mRFP1 (SEQ ID NO:8).

FIG. 25 provides the nucleotide sequence of a modified Discosomawild-type red fluorescent protein open reading frame with humanizedcodon usage (SEQ ID NO:23).

FIG. 26 illustrates the maturation of Q66M DsRed relative to thewild-type DsRed protein.

FIG. 27 shows the excitation and emission spectra of wild-type DsRed(dsRed w.t.) and Q66M DsRed (dsRED Q66M), plotting relative intensity asa function of wavelength.

FIG. 28 shows Coomassie-stained bands on SDS-polyacrylamide gel,representative of wild-type DsRed, Q66M DsRed and K83DsRed afterhydrolysis at pH 1.

FIG. 29 shows the absorption spectrum of mRFP1, mRFP Q66M, and mRFP1.1.The absorption spectrum is normalized to the 280 nm peak which shouldapproximate the total protein concentration. The emission spectrum(measured with excitation at 550 nm) is normalized to its respectiveabsorption maximum for sake of representation.

FIG. 30 provides the amino acid sequence of mRFP1.1 (SEQ ID NO:79).

FIG. 31 provides the nucleotide sequence of mRFP1.1 (SEQ ID NO:80).

FIG. 32 shows a table of properties of DsRed variants, providingexcitation wavelengths and emission wavelengths (as ex/em in nm)extinction coefficients (as M⁻¹cm⁻¹), quantum yield, listing ofmutations with respect to the parent sequence (e.g., ins (DNMA) (SEQ IDNO:111)), ins (NNMA) (SEQ ID NO:112), and comments on the properties ofthe listed DsRed variants.

FIG. 33 provides the amino acid and DNA sequence of dimer2.2MMM (dimer3)(dTomato) (SEQ ID NO: 81 and SEQ ID NO: 82)

FIG. 34 provides the amino acid and DNA sequence of mRFP1.5 (SEQ ID NO:83 and SEQ ID NO: 84).

FIG. 35 provides the amino acid and DNA sequence of OrS4-9 (SEQ ID NO:85 and SEQ ID NO: 86).

FIG. 36 provides the amino acid and DNA sequence of Y13 (mYOFP1.3)(mBanana) (SEQ ID NO. 87 and SEQ ID NO: 88).

FIG. 37 provides the amino acid and DNA sequence of mFRFP (F2Q6)(mGrape2) (SEQ ID NO: 89 and SEQ ID NO: 90).

FIG. 38 provides the amino acid and DNA sequence of mRFP2 (mCherry) (SEQID NO: 92 and SEQ ID NO: 93).

FIG. 39 provides the amino acid and DNA sequence of mOFP (74-11) (SEQ IDNO: 94) and (SEQ ID NO: 95).

FIG. 40 provides the amino acid and DNA sequence of mROFP (A2/6-6) (SEQID NO: 96 and SEQ ID NO: 97).

FIG. 41 provides the amino acid and DNA sequence of mStrawberry (SEQ IDNO: 98 and SEQ ID NO: 99).

FIG. 42 provides the amino acid and DNA sequence of mTangerine (SEQ IDNO: 100 and SEQ ID NO: 101).

FIG. 43 provides the amino acid and DNA sequence of mOrange (MOFP1) (SEQID NO: 102 and SEQ ID NO: 103).

FIG. 44 provides the amino acid and DNA sequence of mHoneydew (SEQ IDNO: 104 and SEQ ID NO: 105).

FIG. 45 provides the amino acid and DNA sequence of tdTomato (SEQ ID NO:106 and SEQ ID NO: 107).

FIG. 46 provides the amino acid and DNA sequence of mGrape1 (SEQ ID NO:108 and SEQ ID NO: 109).

FIG. 47A provides excitation spectra for new RFP variants. Spectra arenormalized to the excitation and emission peak for each protein.Excitation curves are shown as solid or dashed lines for mRFP1 variantsand as a dotted-line for dTomato and tdTomato, with colors correspondingto the color of each variant.

FIG. 47B provides emission spectra for new RFP variants. Spectra arenormalized to the excitation and emission peak for each protein.Emission curves are shown as solid or dashed lines for mRFP1 variantsand as a dotted line for dTomato and tdTomato, with colors correspondingto the color of each variant.

FIG. 48A provides sequence alignment of new mRFP variants with wild-typeDsRed (SEQ ID NO:1) and mRFP1 (SEQ ID NO:8). Internal residues areshaded. mRFP1 mutations are shown in blue, and critical mutations inmCherry (SEQ ID NO:92), mStrawberry (SEQ ID NO:98), mTangerine (SEQ IDNO:100), mOrange (SEQ ID NO:102), mBanana (SEQ ID NO:87), and mHoneydew(SEQ ID NO:104) are shown in colors corresponding to the color of eachvariant. GFP-type termini on new mRFP variants are shown in green.

FIG. 48B provides genealogy of DsRed-derived variants, with mutationscritical to the phenotype of each new variant (e.g., NNMA ins. (SEQ IDNO:112)).

FIG. 49 provides emission spectra for 400 nm excitation for azinc-finger fused with mOrange on its N-terminus and T-Sapphire on itsC-terminus.

FIG. 50 provides mRFP1 and mCherry C-/N-terminal 6×His tag absorbancespectra, illustrating sensitivity to N- and C-terminal fusions.

FIG. 51 provides photobleaching curves for new RFP variants.

FIG. 52 illustrates discrimination of E. coli transfected with sixdifferent fluorescent proteins (FPs).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice the presentinvention. For purposes of the present invention, the following termsare defined.

The term “nucleic acid molecule” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single-strandedor double-stranded form, and, unless specifically indicated otherwise,encompasses polynucleotides containing known analogs of naturallyoccurring nucleotides that can function in a similar manner as naturallyoccurring nucleotides. It will be understood that when a nucleic acidmolecule is represented by a DNA sequence, this also includes RNAmolecules having the corresponding RNA sequence in which “U” (uridine)replaces “T” (thymidine).

The term “recombinant nucleic acid molecule” refers to a non-naturallyoccurring nucleic acid molecule containing two or more linkedpolynucleotide sequences. A recombinant nucleic acid molecule can beproduced by recombination methods, particularly genetic engineeringtechniques, or can be produced by a chemical synthesis method. Arecombinant nucleic acid molecule can encode a fusion protein, forexample, a fluorescent protein variant of the invention linked to apolypeptide of interest. The term “recombinant host cell” refers to acell that contains a recombinant nucleic acid molecule. As such, arecombinant host cell can express a polypeptide from a “gene” that isnot found within the native (non-recombinant) form of the cell.

Reference to a polynucleotide “encoding” a polypeptide means that, upontranscription of the polynucleotide and translation of the mRNA producedtherefrom, a polypeptide is produced. The encoding polynucleotide isconsidered to include both the coding strand, whose nucleotide sequenceis identical to an mRNA, as well as its complementary strand. It will berecognized that such an encoding polynucleotide is considered to includedegenerate nucleotide sequences, which encode the same amino acidresidues. Nucleotide sequences encoding a polypeptide can includepolynucleotides containing introns as well as the encoding exons.

The term “expression control sequence” refers to a nucleotide sequencethat regulates the transcription or translation of a polynucleotide orthe localization of a polypeptide to which to which it is operativelylinked. Expression control sequences are “operatively linked” when theexpression control sequence controls or regulates the transcription and,as appropriate, translation of the nucleotide sequence (i.e., atranscription or translation regulatory element, respectively), orlocalization of an encoded polypeptide to a specific compartment of acell. Thus, an expression control sequence can be a promoter, enhancer,transcription terminator, a start codon (ATG), a splicing signal forintron excision and maintenance of the correct reading frame, a STOPcodon, a ribosome binding site, or a sequence that targets a polypeptideto a particular location, for example, a cell compartmentalizationsignal, which can target a polypeptide to the cytosol, nucleus, plasmamembrane, endoplasmic reticulum, mitochondrial membrane or matrix,chloroplast membrane or lumen, medial trans-Golgi cisternae, or alysosome or endosome. Cell compartmentalization domains are well knownin the art and include, for example, a peptide containing amino acidresidues 1 to 81 of human type II membrane-anchored proteingalactosyltransferase, or amino acid residues 1 to 12 of the presequenceof subunit IV of cytochrome c oxidase (see, also, Hancock et al., EMBOJ. 10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988;U.S. Pat. No. 5,776,689, each of which is incorporated herein byreference).

The term “operatively linked” or “operably linked” or “operativelyjoined” or the like, when used to describe chimeric proteins, refer topolypeptide sequences that are placed in a physical and functionalrelationship to each other. In a most preferred embodiment, thefunctions of the polypeptide components of the chimeric molecule areunchanged compared to the functional activities of the parts inisolation. For example, a fluorescent protein of the present inventioncan be fused to a polypeptide of interest. In this case, it ispreferable that the fusion molecule retains its fluorescence, and thepolypeptide of interest retains its original biological activity. Insome embodiments of the present invention, the activities of either thefluorescent protein or the protein of interest can be reduced relativeto their activities in isolation. Such fusions can also find use withthe present invention. As used herein, the chimeric fusion molecules ofthe invention can be in a monomeric state, or in a multimeric state(e.g., dimeric).

In another example, the tandem dimer fluorescent protein variant of theinvention comprises two “operatively linked” fluorescent protein units.The two units are linked in such a way that each maintains itsfluorescence activity. The first and second units in the tandem dimerneed not be identical. In another embodiment of this example, a thirdpolypeptide of interest can be operatively linked to the tandem dimer,thereby forming a three part fusion protein.

The term “oligomer” refers to a complex formed by the specificinteraction of two or more polypeptides. A “specific interaction” or“specific association” is one that is relatively stable under specifiedconditions, for example, physiologic conditions. Reference to a“propensity” of proteins to oligomerize indicates that the proteins canform dimers, trimers, tetramers, or the like under specified conditions.Generally, fluorescent proteins such as GFPs and DsRed have a propensityto oligomerize under physiologic conditions although, as disclosedherein, fluorescent proteins also can oligomerize, for example, under pHconditions other than physiologic conditions. The conditions under whichfluorescent proteins oligomerize or have a propensity to oligomerize canbe determined using well known methods as disclosed herein or otherwiseknown in the art.

As used herein, a molecule that has a “reduced propensity tooligomerize” is a molecule that shows a reduced propensity to foamstructures with multiple subunits in favor of forming structures withfewer subunits. For example, a molecule that would normally formtetrameric structures under physiological conditions shows a reducedpropensity to oligomerize if the molecule is changed in such a way thatit now has a preference to form monomers, dimers or trimers. A moleculethat would normally form dimeric structures under physiologicalconditions shows a reduced propensity to oligomerize if the molecule ischanged in such a way that it now has a preference to form monomers.Thus, “reduced propensity to oligomerize” applies equally to' proteinsthat are normally dimers and to proteins that are normally tetrameric.

As used herein, the term “non-tetramerizing” refers to protein formsthat produce trimers, dimers and monomers, but not tetramers. Similarly,“non-dimerizing” refers to protein forms that remain monomeric.

As used herein, the term “efficiency of (chromophore) maturation” withreference to a red fluorescent protein (RFP) indicates the percentage ofthe protein that has matured from a species with a green fluorescentprotein (GFP)-like absorbance spectrum to the final RFP absorbancespectrum. Accordingly, efficiency of maturation is determined afterallowing sufficient time for the maturation process to be practically(e.g.>95%) complete. Preferably, the resultant RFP, e.g. DsRed, willcontain at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90%, even more preferably at least about95%, still more preferably at least about 98%, most preferably at leastabout 99% of the red fluorescent species.

As used herein, the term “brightness,” with reference to a fluorescentprotein, is measured as the product of the extinction coefficient (EC)at a given wavelength and the fluorescence quantum yield (QY).

The term “probe” refers to a substance that specifically binds toanother substance (a “target”). Probes include, for example, antibodies,polynucleotides, receptors and their ligands, and generally can belabeled so as to provide a means to identify or isolate a molecule towhich the probe has specifically bound. The term “label” refers to acomposition that is detectable with or without the instrumentation, forexample, by visual inspection, spectroscopy, or a photochemical,biochemical, immunochemical or chemical reaction. Useful labels include,for example, phosphorus-32, a fluorescent dye, a fluorescent protein, anelectron-dense reagent, an enzymes (such as is commonly used in anELISA), a small molecule such as biotin, digoxigenin, or other haptensor peptide for which an antiserum or antibody, which can be a monoclonalantibody, is available. It will be recognized that a fluorescent proteinvariant of the invention, which is itself a detectable protein, cannevertheless be labeled so as to be detectable by a means other than itsown fluorescence, for example, by incorporating a radionuclide label ora peptide tag into the protein so as to facilitate, for example,identification of the protein during its expression and isolation of theexpressed protein, respectively. A label useful for purposes of thepresent invention generally generates a measurable signal such as aradioactive signal, fluorescent light, enzyme activity, and the like,either of which can be used, for example, to quantitate the amount ofthe fluorescent protein variant in a sample.

The term “nucleic acid probe” refers to a polynucleotide that binds to aspecific nucleotide sequence or sub-sequence of a second (target)nucleic acid molecule. A nucleic acid probe generally is apolynucleotide that binds to the target nucleic acid molecule throughcomplementary base pairing. It will be understood that a nucleic acidprobe can specifically bind a target sequence that has less thancomplete complementarity with the probe sequence, and that thespecificity of binding will depend, in part, upon the stringency of thehybridization conditions. A nucleic acid probes can be labeled as with aradionuclide, a chromophore, a lumiphore, a chromogen, a fluorescentprotein, or a small molecule such as biotin; which itself can be bound,for example, by a streptavidin complex, thus providing a means toisolate the probe, including a target nucleic acid molecule specificallybound by the probe. By assaying for the presence or absence of theprobe, one can detect the presence or absence of the target sequence orsub-sequence. The term “labeled nucleic acid probe” refers to a nucleicacid probe that is bound, either directly or through a linker molecule,and covalently or through a stable non-covalent bond such as an ionic,van der Waals or hydrogen bond, to a label such that the presence of theprobe can be identified by detecting the presence of the label bound tothe probe.

The term “polypeptide” or “protein” refers to a polymer of two or moreamino acid residues. The terms apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical analogue of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers. The term “recombinant protein” refers toa protein that is produced by expression of a nucleotide sequenceencoding the amino acid sequence of the protein from a recombinant DNAmolecule.

The term “isolated” or “purified” refers to a material that issubstantially or essentially free from components that normallyaccompany the material in its native state in nature. Purity orhomogeneity generally are determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis, high performanceliquid chromatography, and the like. A polynucleotide or a polypeptideis considered to be isolated when it is the predominant species presentin a preparation. Generally, an isolated protein or nucleicacid-molecule represents greater than 80% of the macromolecular speciespresent in a preparation, often represents greater than 90% of allmacromolecular species present, usually represents greater than 95%, ofthe macromolecular species, and, in particular, is a polypeptide orpolynucleotide that purified to essential homogeneity such that it isthe only species detected when examined using conventional methods fordetermining purity of such a molecule.

The term “naturally-occurring” is used to refer to a protein, nucleicacid molecule, cell, or other material that occurs in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism, including in a virus. A naturally occurring material can be inits form as it exists in nature, and can be modified by the hand of mansuch that, for example, is in an isolated form.

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or antigen-bindingfragments thereof, which specifically bind and recognize an analyte(antigen). The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes. Antibodiesexist as intact immunoglobulins and as well characterizedantigen-binding fragments of an antibody, which can be produced bydigestion with a peptidase or can using recombinant DNA methods. Suchantigen-binding fragments of an antibody include, for example, Fv, Fab′and F(ab)′₂ fragments. The term “antibody,” as used herein, includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies. The term “immunoassay” refers to an assay that utilizesan antibody to specifically bind an analyte. An immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the analyte.

The term “identical,” when used in reference to two or morepolynucleotide sequences or two or more polypeptide sequences, refers tothe residues in the sequences that are the same when aligned for maximumcorrespondence. When percentage of sequence identity is used-inreference to a polypeptide, it is recognized that one or more residuepositions that are not otherwise identical can differ by a conservativeamino acid substitution, in which a first amino acid residue issubstituted for another amino acid residue having similar chemicalproperties such as a similar charge or hydrophobic or hydrophiliccharacter and, therefore, does not change the functional properties ofthe polypeptide. Where polypeptide sequences differ in conservativesubstitutions, the percent sequence identity can be adjusted upwards tocorrect for the conservative nature of the substitution. Such anadjustment can be made using well known methods, for example, scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions can be calculated using any-well knownalgorithm (see, for example, Meyers and Miller, Comp. Appl. Biol. Sci.4:11-17, 1988; Smith and Waterman, Adv. Appl. Math. 2:482, 1981;Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman,Proc. Natl. Acad. Sci., USA 85:2444 (1988); Higgins and Sharp, Gene73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153; 1989; Corpet etal., Nucl. Acids Res. 16:10881-10890, 1988; Huang, et al., Comp. Appl.Biol. Sci. 8:155-165, 1992; Pearson et al., Meth. Mol. Biol.,24:307-331, 1994). Alignment also can be performed by simple visualinspection and manual alignment of sequences.

The term “conservatively modified variation,” when used in reference toa particular polynucleotide sequence, refers to different polynucleotidesequences that encode identical or essentially identical amino acidsequences, or where the polynucleotide does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identicalpolynucleotides encode any given polypeptide. For instance, the codonsCGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.Thus, at every position where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons describedwithout altering the encoded polypeptide. Such nucleotide sequencevariations are “silent variations,” which can be considered a species of“conservatively modified variations.” As such, it will be recognizedthat each polynucleotide sequence disclosed herein as encoding afluorescent protein variant also describes every possible silentvariation. It will also be recognized that each codon in apolynucleotide, except AUG, which is ordinarily the only codon formethionine, and UUG, which is ordinarily the only codon for tryptophan,can be modified to yield a functionally identical molecule by standardtechniques. Accordingly, each silent variation of a polynucleotide thatdoes not change the sequence of the encoded polypeptide is implicitlydescribed herein. Furthermore, it will be recognized that individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids (typically less than 5%,and generally less than 1%) in an encoded sequence can be consideredconservatively modified variations, provided alteration results in thesubstitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitutions providing functionally similaramino acids are well known in the art, including the following sixgroups, each of which contains amino acids that are consideredconservative substitutes for each another:

1) Alanine (Ala, A), Serine (Ser, S), Threo nine (Thr, T);

2) Aspartic acid (Asp, D), Glutamic acid (Glu, E);

3) Asparagine (Asn, N), Glutamine (Gln, Q);

4) Arginine (Arg, R), Lysine (Lys, K);

5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Valine(Val, V); and

6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, V).

Two or more amino acid sequences or two or more nucleotide sequences areconsidered to be “substantially identical” or “substantially similar” ifthe amino acid sequences or the nucleotide sequences share at least 80%sequence identity with each other, or with a reference sequence over agiven comparison window. Thus, substantially similar sequences includethose having, for example, at least 85% sequence identity, at least 90%sequence identity, at least 95% sequence identity, or at least 99%sequence identity.

A subject nucleotide sequence is considered “substantiallycomplementary” to a reference nucleotide sequence if the complement ofthe subject nucleotide sequence is substantially identical to thereference nucleotide sequence. The term “stringent conditions” refers toa temperature and ionic conditions used in a nucleic acid hybridizationreaction. Stringent conditions are sequence dependent and are differentunder different environmental parameters. Generally, stringentconditions are selected to be about 5° C. to 20° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The ^(TM) is the temperature, under defined ionicstrength and pH, at which 50% of the target sequence hybridizes to aperfectly matched probe.

The term “allelic variants” refers to polymorphic forms of a gene at aparticular genetic locus, as well as cDNAs derived from mRNA transcriptsof the genes, and the polypeptides encoded by them. The term “preferredmammalian codon” refers to the subset. of codons from among the set ofcodons encoding an amino acid that are most frequently used in proteinsexpressed in mammalian cells as chosen from the following list: Gly(GGC, GGG); Glu (GAG); Asp (GAC); Val (GUG, GUC); Ala (GCC, GCU); Ser(AGC, UCC); Lys (AAG); Asn (AAC); Met (AUG); Ile (AUC); Thr (ACC); Trp(UGG); Cys (UGC); Tyr (UAU, UAC); Leu (CUG); Phe (UUC); Arg (CGC, AGG,AGA); Gln (CAG); His (CAC); and Pro (CCC).

Fluorescent molecules are useful in fluorescence resonance energytransfer, FRET, which involves a donor molecule and an acceptormolecule. To optimize the efficiency and detectability of FRET between adonor and acceptor molecule, several factors need to be balanced. Theemission spectrum of the donor should overlap as much as possible withthe excitation spectrum of the acceptor to maximize the overlapintegral. Also, the quantum yield of the donor moiety and the extinctioncoefficient of the acceptor should be as high as possible to maximizeRo, which represents the distance at which energy transfer efficiency is50%. However, the excitation spectra of the donor and acceptor shouldoverlap as little as possible so that a wavelength region can be foundat which the donor can be excited efficiently without directly excitingthe acceptor because fluorescence arising from direct excitation of theacceptor can be difficult to distinguish from fluorescence arising fromFRET. Similarly, the emission spectra of the donor and acceptor shouldoverlap as little as possible so that the two emissions can be clearlydistinguished. High fluorescence quantum yield of the acceptor moiety isdesirable if the emission from the acceptor is to be measured either asthe sole readout or as part of an emission ratio. One factor to beconsidered in choosing the donor and acceptor pair is the efficiency offluorescence resonance energy transfer between them. Preferably, theefficiency of FRET between the donor and acceptor is at least 10%, morepreferably at least 50% and even more preferably at least 80%.

The term “fluorescent property” refers to the molar extinctioncoefficient at an appropriate excitation wavelength, the fluorescencequantum efficiency, the shape of the excitation spectrum or emissionspectrum, the excitation wavelength maximum and emission wavelengthmaximum, the ratio of excitation amplitudes at two differentwavelengths, the ratio of emission amplitudes at two differentwavelengths, the excited state lifetime, or the fluorescence anisotropy.A measurable difference in any one of these properties between wild typeAequorea GFP and a spectral variant, or a mutant thereof, is useful. Ameasurable difference can be determined by determining the amount of anyquantitative fluorescent property, e.g., the amount of fluorescence at aparticular wavelength, or the integral of fluorescence over the emissionspectrum. Determining ratios of excitation amplitude or emissionamplitude at two different wavelengths (“excitation amplitude ratioing”and “emission amplitude ratioing”, respectively) are particularlyadvantageous because the ratioing process provides an internal referenceand cancels out variations in the absolute brightness of the excitationsource, the sensitivity of the detector, and light scattering orquenching by the sample.

As used herein, the term “fluorescent protein” refers to any proteinthat can fluoresce when excited with an appropriate electromagneticradiation, except that chemically tagged proteins, wherein thefluorescence is due to the chemical tag, and polypeptides that fluoresceonly due to the presence of certain amino acids such as tryptophan ortyrosine, whose emission peaks at ultraviolet wavelengths (i.e., lessthat about 400 nm) are not considered fluorescent proteins for purposesof the present invention. In general, a fluorescent protein useful forpreparing a composition of the invention or for use in a method of theinvention is a protein that derives its fluorescence fromautocatalytically forming a chromophore. A fluorescent protein cancontain amino acid sequences that are naturally occurring or that havebeen engineered (i.e., variants or mutants). When used in reference to afluorescent protein, the term “mutant” or “variant” refers to a proteinthat is different from a reference protein. For example, a spectralvariant of Aequorea GFP can be derived from the naturally occurring GFPby engineering mutations such as amino acid substitutions into thereference GFP protein. For example ECFP is a spectral variant of GFPthat contains substitutions with respect to GFP (compare SEQ ID NOs: 10and 11).

Many cnidarians use green fluorescent proteins as energy transferacceptors in bioluminescence. The term “green fluorescent protein” isused broadly herein to refer to a protein that fluoresces green light,for example, Aequorea GFP (SEQ ID NO: 10). GFPs have been isolated fromthe Pacific Northwest jellyfish, Aequorea victoria, the sea pansy,Renilla reniformis, and Phialidium gregarium (Ward et al., Photochem.Photobiol. 35:803-808, 1982; Levine et al., Comp. Biochem. Physiol.72B:77-85, 1982, each of which is incorporated herein by reference).Similarly, reference is made herein to “red fluorescent proteins”, whichfluoresce red, “cyan fluorescent proteins,” which fluoresce cyan, andthe like. RFPs, for example, have been isolated from the corallimorphDiscosoma (Matz et al., Nature Biotechnology 17:969-973 [1999]). Theterm “red fluorescent protein,” or “RFP” is used in the broadest senseand specifically covers the Discosoma RFP (DsRed), and red fluorescentproteins from any other species, such as coral and sea anemone, as wellas variants thereof as long as they retain the ability to fluoresce redlight.

The term “coral” as used herein encompasses species within the classAnthozoa, and includes specifically both corals and corallimorphs.

A variety of Aequorea GFP-related fluorescent proteins having usefulexcitation and emission spectra have been engineered by modifying theamino acid sequence of a naturally occurring GFP from A. victoria (seePrasher et al., Gene 111:229-233, 1992; Heim et al., Proc. Natl. Acad.Sci. USA 91:12501-12504, 1994; U.S. Pat. No. 5,625,048; Internationalapplication PCT/US95/14692, now published as PCT WO96/23810, each ofwhich is incorporated herein by reference). As used herein, reference toa “related fluorescent protein” refers to a fluorescent protein that hasa substantially identical amino acid sequence when compared to areference fluorescent protein. In general, a related fluorescentprotein, when compared to the reference fluorescent protein sequence,has a contiguous sequence of at least about 150 amino acids that sharesat least about 85% sequence identity with the reference fluorescentprotein, and particularly has a contiguous sequence of at least about200 amino acids that shares at least about 95% sequence identity withthe reference fluorescent protein. Thus, reference is made herein to an“Aequorea-related fluorescent protein” or to a “GFP-related fluorescentprotein,” which is exemplified by the various spectral variants and GFPmutants that have amino acid sequences that are substantially identicalto A. victoria GFP (SEQ ID NO: 10), to a “Discosoma-related fluorescentprotein” or a “DsRed-related fluorescent related protein,” which isexemplified by the various mutants that have amino acid sequencessubstantially identical to that of DsRed (SEQ ID NO: 1), and the like,for example, a Renilla-related fluorescent protein or aPhialidium-related fluorescent protein.

The term “mutant” or “variant” also is used herein in reference to afluorescent protein that contains a mutation with respect to acorresponding wild type fluorescent protein. In addition, reference ismade herein to a “spectral variant” or “spectral mutant” of afluorescent protein to indicate a mutant fluorescent protein that has adifferent fluorescence characteristic with respect to the correspondingwild type fluorescent protein. For example, CFP, YFP, ECFP (SEQ ID NO:11), EYFP-V68L/Q69K (SEQ ID NO: 12), and the like are GFP spectralvariants.

Aequorea GFP-related fluorescent proteins include, for example, wildtype (native) Aequorea victoria GFP (Prasher et al., supra, 1992; see,also, SEQ ID NO: 10), allelic variants of SEQ ID NO: 10, for example, avariant having a Q80R substitution (Chalfie et al., Science 263:802-805,1994, which is incorporated herein by reference); and spectral variantsof GFP such as CFP, YFP, and enhanced and otherwise modified formsthereof (U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476;5,998,204; and 5,777,079, each of which is incorporated herein byreference), including GFP-related fluorescent proteins having one ormore folding mutations, and fragments of the proteins that arefluorescent, for example, an A. victoria GFP from which the twoN-terminal amino acid residues have been removed. Several of thesefluorescent proteins contain different aromatic amino acids within thecentral chromophore and fluoresce at a distinctly shorter wavelengththan the wild type GFP species. For example, the engineered GFP proteinsdesignated P4 and P4-3 contain, in addition to other mutations, thesubstitution Y66H; and the engineered GFP proteins designated W2 and W7contain, in addition to other mutations, Y66W.

The term “non-tetramerizing fluorescent protein” is used broadly hereinto refer to normally tetrameric fluorescent proteins that have beenmodified such that they have a reduced propensity to tetramerize ascompared to a corresponding unmodified fluorescent protein. As such,unless specifically indicated otherwise, the term “non-tetramerizingfluorescent protein” encompasses dimeric fluorescent proteins, tandemdimer fluorescent proteins, as well as fluorescent proteins that remainmonomeric.

As used herein, the term “aggregation” refers to the tendency of anexpressed protein to form insoluble precipitates or visible punctae andis to be distinguished from “oligomerization”. In particular, mutationsthat reduce aggregation, e.g., increase the solubility of the protein,do not necessarily reduce oligomerization, i.e., convert tetramers todimers or monomers.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides fluorescent protein variants that can bederived from fluorescent proteins that have a propensity to dimerize ortetramerize. As disclosed herein, in one embodiment of the invention, afluorescent protein variant of the invention can be derived from anaturally occurring fluorescent protein or from a spectral variant ormutant thereof, and contains at least one mutation that reduces oreliminates the propensity of the fluorescent protein to oligomerize. Inparticular, the present invention provides dimeric and monomeric redfluorescent proteins (RFP) and RFP variants with reduced propensity tooligomerize. As disclosed herein, in a further embodiment of theinvention, a fluorescent protein is provided having improved efficacy ofmaturation. In particular, the present invention provides dimeric andmonomeric red fluorescent proteins (RFP) and RFP variants with improvedefficacy of maturation. In embodiments of the invention, fluorescentprotein variants are provided which contain at least one mutation thatreduces or eliminates the propensity of the fluorescent protein tooligomerize and which contain at least one mutation that improves theefficacy of maturation of fluorescence in the protein variant—ascompared to other variants including the parent protein.

The cloning of a red fluorescent protein from Discosoma (DsRed) raised agreat deal of interest due to its tremendous potential as a tool for theadvancement of cell biology. However, a careful investigation of theproperties of this protein revealed several problems that would precludeDsRed from being as widely accepted as the Aequorea GFP and its blue,cyan, and yellow variants, which have found widespread use as bothgenetically encoded indicators for tracking gene expression and asdonor/acceptor pairs for fluorescence resonance energy transfer (FRET).Extending the spectrum of available colors to red wavelengths wouldprovide a distinct new label for multicolor tracking of fusion proteinsand together with GFP would provide a new FRET donor/acceptor pair thatwould be superior to the currently preferred cyan/yellow pair.

The three most pressing problems with the 28 kDa DsRed are its strongtendency to oligomerize, its slow maturation, and its inefficientmaturation from a species with a GFP-like spectrum to the ultimate RFPspectrum.

A variety of techniques have been used to determine that DsRed is anobligate tetramer both in vitro and in vivo. For numerous reasons, theoligomeric state of DsRed is problematic for applications in which it isfused to a protein of interest in order to monitor trafficking orinteractions of the latter. Using purified protein, it was shown thatDsRed requires greater than 48 hours to reach >90% of its maximal redfluorescence (see below). During the maturation process, a greenintermediate initially accumulates and is slowly converted to the finalred form. However, the conversion of the green component does notproceed to completion and thus a fraction of aged DsRed remains green.The primary disadvantage of the incomplete maturation is an excitationspectrum that extends well into the green wavelengths due to energytransfer between the green and red species within the tetramer. This isa particularly serious problem due to overlap with the excitationspectra of potential FRET partners such as GFP.

The original report of the cloning of DsRed provided an in vivoapplication marking the fates of Xenopus blastomeres after 1 week ofdevelopment (Matz et al., Nature Biotechnology 17:969-973 [1999]). Asdisclosed herein, DsRed has been characterized with respect to the timethe red fluorescence takes to appear, the pH sensitivity of thechromophore, how strongly the chromophore absorbs light and fluoresces,how readily the protein photobleaches, and whether the protein normallyexists as a monomer or an oligomer in solution. The results demonstratethat DsRed provides a useful complement to or alternative for GFP andits spectral mutants. In addition, DsRed mutants that arenon-fluorescent or that are blocked or slowed in converting from greento red emission were characterized, including mutants in which theeventual fluorescence is substantially red-shifted from wild type DsRed(see, Baird et al., Proc. Natl. Acad. Sci., USA 97:11984-11989, 2000;Gross et al., Proc. Natl. Acad. Sci., USA 97:11990-11995, 2000, each ofwhich is incorporated herein by reference).

Red Fluorescent Protein Variants with Improved Efficacy of Maturation

The present invention provides RFP variants that show more efficientchromophore maturation than a reference wild-type or variant RFP, as aresult of at least one amino acid alteration within the reference(wild-type or variant) sequence.

The wild-type RFP protein typically consists of about 70% red proteinwith about 30% contamination by the green, immature form of the protein.Through careful mass spectrometric and biochemical investigations, ithas been determined that the Cα-N bond of Q66 in DsRed is oxidized asthe protein matures into its red form, which, in turn, led to a furtherinvestigation of the role of the amino acid at-position 66 as it relatesto chromophore maturation. By site-directed mutagenesis, it wasdetermined that the substitution of methionine (M) for the nativeglutamine (Q) at amino acid position 66 yielded a protein that showed adeeper pink color than the wild-type protein, and contained less of theimmature green form than wild-type DsRed. In addition, the Q66M DsRedvariant was found to mature more quickly than wild-type DsRed.Additional experiments have shown that the Q66M mutation retained itsadvantageous properties, also when introduced into non-tetramerizing,i.e. dimeric or monomeric DsRed variants, which contained additionalmutations. Further details of these findings are set forth in theExamples below: Thus, an RFP variant of the invention can be derivedfrom—a naturally occurring (wild-type) RFP or from a spectral variant ormutant thereof, and contains at least one mutation that makeschromophore maturation more efficient.

While the invention is illustrated with reference to the Q66M mutationin DsRed, it will be understood that it is not so limited. Mutations ofother amino acids within the wild-type DsRed sequence that play a rolein chromophore structure, orientation and/or maturation can also yieldDsRed variants with improved maturation efficiency. Similarly, aminoacid alterations (e.g. substitutions) at corresponding (homologous)positions or regions in other RFPs can produce RFP variants showing animprovement in maturation efficiency. All of such variants, alone or incombination with other mutations (substitutions, insertions and/ordeletions) within the wild-type RFP sequence, are specifically withinthe scope of the invention. Thus an additional exemplary amino acidalteration that is believed to improve maturation of both the wild-typeDsRed protein and DsRed variants, including Q66M DsRed is a substitutionat amino acid position 147 of wild-type DsRed. A preferred substitutionat this position is T147S, but other substitutions resulting in similarimprovements in spectral properties and, in particular, in theefficiency and potentially speed of maturation, are also possible.Especially, substitution of amino acids with similar properties ofthreonine (T) are expected to yield such variants.

In a specific embodiment, the invention concerns RFP variants withimproved maturation efficiency that have a reduced propensity totetramerize, as a result of one or more further mutations within the RFPmolecule. In particular, in this embodiment, the invention concernsnon-tetramerizing, such as dimeric or monomeric, DsRed variants thatshow enhanced maturation efficiency relative to the correspondingnon-tetramerizing DsRed variant. Further details about the design andpreparation of such variants are provided below.

In brief, the RFP variants of the invention can be derived from RFPsthat have a propensity to dimerize or tetramerize. As disclosed herein,an RFP variant of the invention can be derived from a naturallyoccurring RFP or from a spectral variant or mutant thereof, and containsat least one mutation that enhances maturation efficiency, andoptionally at least one additional mutation that reduces or eliminatesthe propensity of the RFP to oligomerize.

A fluorescent protein variant of the invention can be derived from anyfluorescent protein that is known to oligomerize, including, forexample, a green fluorescent protein (GFP) such as an Aequorea victoriaGFP (SEQ ID NO: 10), a Renilla reniformis GFP, a Phialidium gregariumGFP; a red fluorescent protein (RFP) such as a Discosoma RFP (SEQ ID NO:1); or a fluorescent protein related to a GFP or an RFP. Thus, thefluorescent protein can be a cyan fluorescent protein (CFP), a yellowfluorescent protein (YFP), an enhanced GFP (EGFP; SEQ ID NO: 13), anenhanced CFP (ECFP; SEQ ID NO: 11), an enhanced YFP (EYFP; SEQ ID NO:15), a DsRed fluorescent protein (SEQ ID NO: 1), a homologue in anyother species, or a mutant or variant of such fluorescent proteins.

As disclosed herein, the propensity of the fluorescent protein variantof the invention to oligomerize is reduced or eliminated. There are twobasic approaches to reduce the propensity of the fluorescent protein,e.g., RFP such as DsRed, to form intermolecular oligomers, (1)oligomerization can be reduced or eliminated by introducing mutationsinto appropriate regions of the fluorescent protein, e.g., an RFPmolecule, and (2) two subunits of the fluorescent protein canoperatively link, e.g., link RFP to each other by a linker, such as apeptide linker. If oligomerization is reduced or eliminated by followingapproach (1), it is usually necessary to introduce additional mutationsinto the molecule, in order to restore fluorescence, which is typicallylost or greatly impaired as a result of introducing mutations at theoligomer interfaces.

Red Fluorescent Protein Variants with Reduced Propensity to Oligomerize

The present invention provides fluorescent protein variants where thedegree of oligomerization of the fluorescent protein is reduced oreliminated by the introduction of amino acid substitutions to reduce orabolish the propensity of the constituent monomers to tetramerize. Inone embodiment, the resulting structures have a propensity to dimerize.In other embodiments, the resulting structures have a propensity toremain monomeric.

Various dimer forms can be created. For example, an AB orientation dimercan be formed, or alternatively, an AC orientation dimer can be formed.However, with the creation of dimeric forms, fluorescence or the rate ofmaturation of fluorescence, can be lost. The present invention providesmethods for the generation of dimeric forms that display detectablefluorescence, and furthermore, fluorescence that has advantageous ratesof maturation.

In one embodiment, the dimer is an intermolecular dimer. Furthermore,the dimer can be a homodimer (comprising two molecules of the identicalspecies) or a heterodimer (comprising two molecules of differentspecies). In a preferred embodiment, dimers will spontaneously form inphysiological conditions. As used herein, the molecules that form suchtypes of structures are said to have a reduced tendency to oligomerize,as the monomeric units have reduced or non-existent ability to formtetrameric intermolecular oligomers.

A non-limiting, illustrative example of such a dimeric red fluorescentprotein variant is described herein, and is termed “dimer2.” The dimer2nucleotide sequence is provided in SEQ ID NO: 7 and FIG. 21. The dimer2polypeptide is provided in SEQ ID NO: 6 and FIG. 22.

In an attempt to produce a still further advantageous form of the DsRedvariant dimer, a novel strategy to synthesize a “tandem” DsRed variantdimer was devised. This approach utilized covalent tethering of twoengineered monomeric DsRed units to yield a dimeric form of DsRed withadvantageous properties. The basic strategy was to fuse two copies of anAC dimer with a polypeptide linker such that the critical dimerinteractions could be satisfied through intramolecular contacts with thetandem partner encoded within the same polypeptide. Such operably linkedhomodimers or heterodimers are referred to herein as “tandem dimers,”and have a substantially reduced propensity to form tetramericstructures.

Illustrative examples of such tandem red fluorescent protein variantdimers include, without limitation, two monomeric units of the dimer2species (SEQ ID NO: 6) operably covalently linked by a peptide linker,preferably about 9 to about 25, more preferably about 9 to 20 amino acidresidues in length. Such linkers finding use with the invention include,but are not limited to, for example, the 9 residue linker RMGTGSGQL (SEQID NO: 16), the 12 residue linker GHGTGSTGSGSS (SEQ ID NO: 17), the 13residue linker RMGSTSGSTKGQL (SEQ ID NO: 18), or the 22 residue linkerRMGSTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19). As noted above, the subunits ofsuch tandem dimers preferably contain mutations relative to thewild-type DsRed sequence of SEQ ID NO: 1, in order to preserve/restorefluorescent properties. An illustrative example of the tandem redfluorescent protein dimers herein is a dimer composed of two monomers,wherein at least one of the monomers is a variant DsRed, which has anamino acid sequence of SEQ ID NO: 6, operatively linked by a peptidelinker, preferably about 9 to about 25, more preferably about 10 toabout 20 amino acid residues in length, including any of the 9, 12, 13,and 22 residue linkers above. Yet another illustrative example of atandem red fluorescent protein dimer herein is a tandem dimer composedof two identical or different DsRed variant monomeric subunits at leastone of which contains, for example, the following substitutions withinthe DsRed polypeptide of SEQ ID NO: 1: N42Q, V44A, V71A, F118L, K163Q,S179T, S197T, T217S (mutations internal to the β-barrel); R2A, K5E andN6D (aggregation reducing mutations); I125R and V127T (AB interfacemutations); and T21S, H41T, C117T and S131P (miscellaneous surfacemutations). Just as in the other illustrative dimers, the two monomericsubunits may be fused by a peptide linker, preferably about 9 to about25, more preferably about 10 to about 25 amino acid residues in length,such as any of the 9, 12, 13, and 22 residue linkers above. Shorterlinkers are generally preferable to longer linkers, as long as they donot significantly slow affinity maturation or otherwise interfere withthe fluorescent and spectral properties of the dimer. As noted above,the two monomeric subunits within a dimer may be identical or different.Thus, for example, one subunit may be the wild-type DsRed monomer of SEQID NO: 1 operatively linked to a variant DsRed polypeptide, such as anyof the DsRed variants listed above or otherwise disclosed herein. Themonomers should be linked such that the critical dimer interactions aresatisfied through intramolecular contacts with the tandem partner. Thepeptide linkers are preferably protease resistant. The peptide linkersspecifically disclosed herein are only illustrative. One skilled in theart will understand that other peptide linkers, preferably proteaseresistant linkers, are also suitable for the purpose of the presentinvention. See, e.g., Whitlow et al., Protein Eng 6:989-995 (1993).

In one embodiment, disclosed in more detail in the examples below, anovel approach was used to overcome the intermolecular oligomerizationpropensity of wild-type DsRed by linking the C-terminus of the A subunitto the N-terminus of the B subunit through a flexible linker to producetandem dimers. Based on the crystal structure of DsRed tetramer, a 10 to20 residue linkers, such as an 18 residue linker (Whitlow et al., Prot.Eng. 6:989-995, 1993, supra, which is incorporated herein by reference)was predicted to be long enough to extend from the C-terminus of the Asubunit to the N-terminus of the C subunit (about 30 Å), but not to theN-terminus of the B subunit (greater than 70 Å). As such,‘oligomerization’ in the tandem dimers is intramolecular, i.e., thetandem dimer of DsRed (tDsRed), for example, is encoded by a singlepolypeptide chain. Furthermore, a combination of tDsRed with the I125Rmutant (tDsRed-I125R) resulted in another dimeric red fluorescentprotein. It should be recognized that this strategy can be generallyapplied to any protein system in which the distance between theN-terminus of one protein and the C-terminus of a dimer partner isknown, such that a linker having the appropriate length can be used tooperatively link the monomers. In particular, this strategy can beuseful for other modifying other fluorescent proteins that haveinteresting spectral properties, but form obligate dimers that aredifficult to disrupt using the targeted mutagenesis method disclosedherein.

Mutagenesis Strategy to Produce Dimeric and Monomeric Red FluorescentProteins

The present invention provides variant fluorescent proteins that have areduced propensity to form tetrameric oligomers (i.e., the propensity toform tetramers is reduced or eliminated) due to the presence of one ormore mutations in the fluorescent protein. As disclosed herein,mutations were introduced into DsRed, and DsRed mutants having reducedoligomerization activity were identified, including, for example, aDsRed-I125R mutant of DsRed of SEQ ID NO: 20. The strategy for producingthe DsRed mutants involved introducing mutations in DsRed that werepredicted to interfere with the dimer interfaces (A-B or A-C, see FIGS.1 and 2) and thus prevent formation of the tetramer. This strategyresulted in the production of DsRed mutants that had a reducedpropensity to form tetramers by disrupting the A-B interface, forexample, using the single replacement of isoleucine 125 with an arginine(I125R).

The basic strategy for decreasing the oligomeric state of DsRed was toreplace key dimer interface residues with charged amino acids,preferably arginine. It is contemplated that dimer formation wouldrequire the targeted residue to interact with the identical residue ofthe dimer partner through symmetry. The resulting high energetic cost ofplacing two positive charges in close proximity should disrupt theinteraction. Initial attempts to break apart the DsRed AC interface (seeFIG. 2A) with the single mutations T147R, H162R, and F224R, consistentlygave non-fluorescent proteins. The AB interface however, proved somewhatless resilient and could be broken with the single mutation I125R togive a poorly red fluorescent dimer that suffered from an increasedgreen component and required more than 10 days to fully mature.

Illustrative examples of mutations (amino acid substitutions) which canfurther improve the fluorescent properties of I125R include mutations inat least one of amino acid positions 163, 179 an 217 within SEQ IDNO: 1. In a preferred embodiment, the I125R variant comprises at leastone of the K163Q/M, S179T and T217S substitutions. Further illustrativevariants may contain additional mutations at position N42 and/or C44within SEQ ID NO: 1. Yet another group of illustrative DsRed dimerscomprise additional mutations at least one of residues I161 and S197within SEQ ID NO: 1. Specific examples of DsRed variants obtained bythis mutagenesis approach include DsRed-I125R, S179T, T217A, andDsRed-I125R, K163Q, T217A, and others (see, e.g., the Examples below).

It is noted that there exists an inconsistency in the naming conventionof the DsRed subunits in the prior art. As shown in FIG. 1, oneconvention assigns the A-B-C-D subunits as shown. However, a differentconvention is also recognized, which is shown in FIG. 2. When viewingthe model of FIG. 1, the AC interface of that figure is equivalent tothe AB interface shown in FIG. 2A. With the exception of FIG. 1,reference to subunit interfaces in the present application is accordingto the convention used in FIG. 2.

A similar directed mutagenesis strategy starting from T1-I125R (see FIG.10A, library D1) was undertaken and eventually identified dimer1. Dimer1was somewhat better than wt DsRed both in terms of brightness and rateof maturation but had a substantial green peak equivalent to that of T1.Dimer1 was also somewhat blue-shifted with an excitation maximum at 551nm and an emission maximum at 579 nm. Error prone PCR on dimer1 (FIG.10A, library D2) resulted in the discovery of dimer1.02 containing themutation V71A in the hydrophobic core of the protein and effectively nogreen component in the excitation spectra. A second round of randommutagenesis (FIG. 10A, library D3) identified the mutations K70R whichfurther decreased the green excitation, S197A which red-shifted thedimer back to DsRed wavelengths and T217S which greatly improved therate of maturation. Unfortunately, K70R and S197A matured relativelyslowly and T217S had a green excitation peak equivalent to DsRed. Usingdimer1.02 as the template, two more rounds of directed mutagenesis wereperformed; the first focusing on the three positions identified above(FIG. 11A, library D3) and the second on C117, F118, F124, and V127(FIG. 10A, library D4).

Continuing with the directed evolution strategy for a total of 4generations, an optimal dimeric variant was produced, which wasdesignated dimer2 (illustrated in FIG. 2B). This variant contains 17mutations, of which eight are internal to the β-barrel (N42Q, V44A,V71A, F118L, K163Q, S179T, S197T and T217S), three are the aggregationreducing mutations found in T1 (R2A, K5E and N6D and see Bevis andGlick, Nat. Biotechnol., 20:83-87 [2002]; and Yanushevich et al., FEBSLett., 511:11-14 [2002]), two are AB interface mutations (I125R andV127T), and 4 are miscellaneous surface mutations (T21S, H41T, C117T andS131P). The dimer2 nucleotide sequence is provided in SEQ ID NO: 7 andFIG. 21. The dimer2 polypeptide is provided in SEQ ID NO: 6 and FIG. 22.

A product of the mutagenesis approach described above is a monomeric redfluorescent protein, designated mRFP1, which contains the followingmutations within the wild-type DsRed sequence of SEQ ID NO: 1: N42Q,V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T,S197I, T217A, R2A, K5E, N6D, I125R, V127T; I180T, R153E, H162K, A164R;L174D, Y192A, Y194K, H222S, L223T, F224G, L225A, T21S, H41T, C117E, andV156A. Of these, the first 13 mutations are internal to the β-barrel. Ofthe remaining 20 external mutations, 3 are aggregation reducingmutations (R2A, K5E, and N6D), 3 are AB interface mutations (I125R,V127T, and I180T), 10 are AC interface mutations (R153E, H162K, A164R,L174D, Y192A, Y194K, H222S, L223T, F224G, and L225A), and 4 areadditional beneficial mutations (T21S, H41T, C117E, and V156A). ThemRFP1 nucleotide sequence is provided in SEQ ID NO: 9 and FIG. 23. ThemRFP1 polypeptide is provided in SEQ ID NO: 8 and FIG. 24.

Other variants, including variants related to mRFP1, may also beproduced by such methods (see, e.g., the Examples below). Thus, althoughmRFP1 is believed to be optimized in many aspects, a person skilled inthe art will appreciate that other mutations within these and otherregions of the wild-type DsRed amino acid sequence (SEQ ID NO: 1) mayalso yield monomeric DsRed variants retaining the qualitative redfluorescing properties of the wild-type DsRed protein. Accordingly,mRFP1 serves merely as an illustration, and embodiments of the inventionare by no means intended to be limited to this particular monomer.

For example, the monomeric DsRed variants herein, e.g. mRFP1, can befurther modified to alter the spectral and/or fluorescent properties ofDsRed. For example, based upon experience with GFP, it is known that inthe excited state, electron density tends to shift from the phenolatetowards the carbonyl end of the chromophore. Therefore, placement ofincreasing positive charge near the carbonyl end of the chromophoretends to decrease the energy of the excited. state and cause a red-shiftin the absorbance and emission wavelength maximum of the protein.Decreasing a positive charge near the carbonyl end of the chromophoretends to have the opposite effect, causing a blue-shift in the protein'swavelengths. Similarly, mutations have been introduced into DsRed toproduce mutants having altered fluorescence characteristics.

Amino acids with charged (ionized D, E, K, and R), dipolar (H, N, Q, S,T, and uncharged D, E and K), and polarizable side groups (e.g., C, F,H, M, W and Y) are useful for altering the ability of fluorescentproteins to oligomerize, especially when they substitute an amino acidwith an uncharged, nonpolar or non-polarizable side chain.

Similarly, monomers of other oligomerizing fluorescent proteins can alsobe prepared following a similar mutagenesis strategy, as illustrated inthe Examples below, and these and other fluorescent protein monomers areintended to be within the scope of the present invention.

Variant Anthozoan Fluorescent Proteins

It is contemplated that the mutagenesis methods provided by the presentinvention can be used to generate advantageous fluorescent proteinvariants that have reduced ability to oligomerize (i.e., tetramerize),and also find uses analogous to the uses of the Discosoma DsRed variantproteins. It is known in the art that the DsRed protein is a member of afamily of highly related homologous proteins sharing high degrees ofamino acid identity and protein structure (see, e.g., Labas et al.,Proc. Natl. Acad. Sci. USA 99:4256-4261 [2002]; and Yanushevich et al.,FEBS Letters 511:11-14 [2002]). These alternative fluorescent proteinsare additionally advantageous since they have the ability to fluoresceat different wavelengths than does Discosoma DsRed. If dimeric ormonomeric forms of these proteins can be produced, they will have greatexperimental potential as fluorescent markers.

Anthozoan species from which related fluorescent proteins have beenidentified include, but are not limited to, Anemonia sp., Clavulariasp., Condylactis sp., Heteractis sp., Renilla sp., Ptilosarcus sp.,Zoonthus sp., Scolymia sp., Montastraea sp., Ricordea sp., Gonioparasp., and others.

Fusion Proteins Comprising the Tandem Dimers and Monomers

Fluorescent proteins fused to target proteins can be prepared, forexample using recombinant DNA methods, and used as markers to identifythe location and amount of the target protein produced. Accordingly, thepresent invention provides fusion proteins comprising a fluorescentprotein variant moiety and a polypeptide of interest. The polypeptide ofinterest can be of any length, for example, about 15 amino acidresidues, about 50 residues, about 150 residues, or up to about 1000amino acid residues or more, provided that the-fluorescent proteincomponent of the fusion protein can fluoresce or can be induced tofluoresce when exposed to electromagnetic radiation of the appropriatewavelength. The polypeptide of interest can be, for example, a peptidetag such as a polyhistidine sequence, a c-myc epitope, a FLAG epitope,and the like; can be an enzyme, which can be used to effect a functionin a cell expressing a fusion protein comprising the enzyme or toidentify a cell containing the fusion protein; can be a protein to beexamined for an ability to interact with one or more other proteins in acell, or any other protein as disclosed herein or otherwise desired.

As disclosed herein, the Discosoma (coral) red fluorescent protein,DsRed, can be used as a complement to or alternative for a GFP orspectral variant thereof. In particular, the invention encompassesfusion proteins of any of the tandem dimeric and monomeric DsRedfluorescent proteins discussed above, and variants thereof, which hasaltered spectral and/or fluorescent characteristics.

A fusion protein, which includes a fluorescent protein variantoperatively linked to one or more polypeptides of interest also isprovided. The polypeptides of the fusion protein can be linked throughpeptide bonds, or the fluorescent protein variant can be linked to thepolypeptide of interest through a linker molecule. In one embodiment,the fusion protein is expressed from a recombinant nucleic acid moleculecontaining a polynucleotide encoding a fluorescent protein variantoperatively linked to one or more polynucleotides encoding one or morepolypeptides of interest.

A polypeptide of interest can be any polypeptide, including, forexample, a peptide tag such as a polyhistidine peptide, or a cellularpolypeptide such as an enzyme, a G-protein, a growth factor receptor, ora transcription factor; and can be one of two or more proteins that canassociate to form a complex. In one embodiment, the fusion protein is atandem fluorescent protein variant construct, which includes a donorfluorescent protein variant, an acceptor fluorescent protein variant,and a peptide linker moiety coupling said donor and said acceptor,wherein cyclized, amino acids of the donor emit light characteristic ofsaid donor, and wherein the donor and the acceptor exhibit fluorescenceresonance energy transfer when the donor is excited, and the linkermoiety does not substantially emit light to excite the donor. As such, afusion protein of the invention can include two or more operativelylinked fluorescent protein variants, which can be linked directly orindirectly, and can further comprise one or more polypeptides ofinterest.

Preparation of DsRed Dimers and Monomers

The present invention also provides polynucleotides encoding fluorescentprotein variants, where the protein can be a dimeric fluorescentprotein, a tandem dimeric fluorescent protein, a monomeric protein, or afusion protein comprising a fluorescent protein operatively linked toone or more polypeptides of interest. In the case of the tandem dimerthe entire dimer may be encoded by one polynucleotide molecule. If thelinker is a non-peptide linker, the two subunits will be encoded byseparate polynucleotide molecules, produced separately, and subsequentlylinked by methods known in the art.

The invention further concerns vectors containing such polynucleotides,and host cell containing a polynucleotide or vector. Also provided is arecombinant nucleic acid molecule, which includes at least onepolynucleotide encoding a fluorescent protein variant operatively linkedto one or more other polynucleotides. The one or more otherpolynucleotides can be, for example, a transcription regulatory elementsuch as a promoter or polyadenylation signal sequence, or a translationregulatory element such as a ribosome binding site. Such a recombinantnucleic acid molecule can be contained in a vector, which can be anexpression vector, and the nucleic acid molecule or the vector can becontained in a host cell.

The vector generally contains elements required for replication in aprokaryotic or eukaryotic host system or both, as desired. Such vectors,which include plasmid vectors and viral vectors such as bacteriophage,baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semlikiforest virus and adeno-associated virus vectors, are well known and canbe purchased from a commercial source (Promega, Madison Wis.;Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or can beconstructed by one skilled in the art (see; for example, Meth. Enzymol.,Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. GeneTher. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993;Kirshenbaum et al.; J. Clin. Invest. 92:381-387, 1993; each of which isincorporated herein by reference).

A vector for containing a polynucleotide encoding a fluorescent proteinvariant can be a cloning vector or an expression vector, and can be aplasmid vector, viral vector, and the like. Generally, the vectorcontains a selectable marker independent of that encoded by apolynucleotide of the invention, and further can contain transcriptionor translation regulatory elements, including a promoter sequence, whichcan provide tissue specific expression of a polynucleotide operativelylinked thereto, which can, but need not, be the polynucleotide encodingthe fluorescent protein variant, for example, a tandem dimer fluorescentprotein, thus providing a means to select a particular cell type fromamong a mixed population of cells containing the introduced vector andrecombinant nucleic acid molecule contained therein.

Where the vector is a viral vector, it can be selected based on itsability to infect one or few specific cell types with relatively highefficiency. For example, the viral vector also can be derived from avirus that infects particular cells of an organism of interest, forexample, vertebrate host cells such as mammalian host cells. Viralvectors have been developed for use in particular host systems,particularly mammalian systems and include, for example, retroviralvectors, other lentivirus vectors such as those based on the humanimmunodeficiency virus. (HIV), adenovirus vectors, adeno-associatedvirus vectors, herpesvirus vectors, vaccinia virus vectors, and the like(see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al.,Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242;1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which isincorporated herein by reference).

Recombinant production of a fluorescent protein variant, which can be acomponent of a fusion protein, involves expressing a polypeptide encodedby a polynucleotide. A polynucleotide encoding the fluorescent proteinvariant is a useful starting material. Polynucleotides encodingfluorescent protein are disclosed herein or otherwise known in the art,and can be obtained using routine methods, then can be modified suchthat the encoded fluorescent protein lacks a propensity to oligomerize.For example, a polynucleotide encoding a GFP can be isolated by PCR ofcDNA from A. victoria using primers based on the DNA sequence ofAequorea GFP (SEQ ID NO: 21). A polynucleotide encoding the redfluorescent protein from Discosoma (DsRed) can be similarly isolated byPCR of cDNA of the Discosoma coral, or obtained from the commerciallyavailable DsRed2 or HcRed1 (CLONTECH). PCR methods are well known androutine in the art (see, for example, U.S. Pat. No. 4,683,195; Mullis etal., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; Erlich, ed.,“PCR Technology” (Stockton Press, NY, 1989)). A variant form of thefluorescent protein then can be made by site-specific mutagenesis of thepolynucleotide encoding the fluorescent protein. Similarly, a tandemdimer fluorescent protein can be expressed from a polynucleotideprepared by PCR or obtained otherwise, using primers that can encode,for example, a peptide linker, which operatively links a first monomerand at least a second monomer of a fluorescent protein.

The construction of expression vectors and the expression of apolynucleotide in transfected cells involves the use of molecularcloning techniques also well known in the art (see Sambrook et al., In“Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor LaboratoryPress 1989); “Current Protocols in Molecular Biology” (eds., Ausubel etal.; Greene Publishing Associates, Inc., and John Wiley & Sons, Inc.1990 and supplements). Expression vectors contain expression controlsequences operatively linked to a polynucleotide sequence of interest,for example, that encodes a fluorescent protein variant, as indicatedabove. The expression vector can be adapted for function in prokaryotesor eukaryotes by inclusion of appropriate promoters, replicationsequences, markers, and the like. An expression vector can betransfected into a recombinant host cell for expression of a fluorescentprotein variant, and host cells can be selected, for example, for highlevels of expression in order to obtain a large amount of isolatedprotein. A host cell can be maintained in cell culture, or can be a cellin vivo in an organism. A fluorescent protein variant can be produced byexpression from a polynucleotide encoding the protein in a host cellsuch as E. coli. Aequorea GFP-related fluorescent proteins, for example,are best expressed by cells cultured between about 15° C. and 30° C.,although higher temperatures such as 37° C. can be used. Aftersynthesis; the fluorescent proteins are stable at higher temperaturesand can be used in assays at such temperatures.

An expressed fluorescent protein variant, which can be a tandem dimerfluorescent protein or a non-oligomerizing monomer, can be operativelylinked to a first polypeptide of interest, further can be linked to asecond polypeptide of interest, for example, a peptide tag, which can beused to facilitate isolation of the fluorescent protein variant,including any other polypeptides linked thereto. For example, apolyhistidine tag containing, for example, six histidine residues, canbe incorporated at the N-terminus or C-terminus of the fluorescentprotein variant, which then can be isolated in a single step usingnickel-chelate chromatography. ‘Additional peptide tags, including ac-myc peptide, a FLAG epitope, or any ligand (or cognate receptor),including any peptide epitope (or antibody, or antigen binding fragmentthereof, that specifically binds the epitope are well known in the artand similarly can be used. (see, for example, Hopp et al., Biotechnology6:1204 (1988); U.S. Pat. No. 5,011,912, each of which is incorporatedherein by reference).

Kits of the Invention

The present invention also provides kits to facilitate and/orstandardize use of compositions provided by the present invention, aswell as facilitate the methods of the present invention. Materials andreagents to carry out these various methods can be provided in kits tofacilitate execution of the methods. As used herein, the term “kit” isused in reference to a combination of articles that facilitate aprocess, assay, analysis or manipulation.

Kits can contain chemical reagents (e.g., polypeptides orpolynucleotides) as well as other components. In addition, kits of thepresent invention can also include, for example but not limited to,apparatus and reagents for sample collection and/or purification,apparatus and reagents for product collection and/or purification,reagents for bacterial cell transformation, reagents for eukaryotic celltransfection, previously transformed or transfected host cells, sampletubes, holders, trays, racks, dishes, plates, instructions to the kituser, solutions, buffers or other chemical reagents, suitable samples tobe used for standardization, normalization, and/or control samples. Kitsof the present invention can also be packaged for convenient storage andsafe shipping, for example, in a box having a lid.

In some embodiments, for example, kits of the present invention canprovide a fluorescent protein of the invention, a polynucleotide vector(e.g., a plasmid) encoding a fluorescent protein of the invention,bacterial cell strains suitable for propagating the vector, and reagentsfor purification of expressed fusion proteins. Alternatively, a kit ofthe present invention can provide the reagents necessary to conductmutagenesis of an Anthozoan fluorescent protein in order to generate aprotein variant having a reduced propensity to oligomerize.

A kit can contain one or more compositions of the invention, forexample, one or a plurality of fluorescent protein variants, which canbe a portion of a fusion protein, or one or a plurality ofpolynucleotides that encode the polypeptides. The fluorescent proteinvariant can be a mutated fluorescent protein having a reduced propensityto oligomerize, such as a non-oligomerizing monomer, or can be a tandemdimer fluorescent protein and, where the kit comprises a plurality offluorescent protein variants, the plurality can be a plurality of themutated fluorescent protein variants, or of the tandem dimer fluorescentproteins, or a combination thereof.

A kit having features of the invention also can contain one or aplurality of recombinant nucleic acid molecules, which encode, in part,fluorescent protein variants, which can be the same or different, andcan further include, for example, an operatively linked secondpolynucleotide containing or encoding a restriction endonucleaserecognition site or a recombinase recognition site, or any polypeptideof interest. In addition, the kit can contain instructions for using thecomponents of the kit, particularly the compositions of the inventionthat are contained in the kit.

Such kits can be particularly useful where they provide a plurality ofdifferent fluorescent protein variants because the artisan canconveniently select one or more proteins having the fluorescentproperties desired for a particular application. Similarly, a kitcontaining a plurality of polynucleotides encoding different fluorescentprotein variants provides numerous advantages. For example, thepolynucleotides can be engineered to contain convenient restrictionendonuclease or recombinase recognition sites, thus facilitatingoperative linkage of the polynucleotide to a regulatory element or to apolynucleotide encoding a polypeptide of interest or, if desired, foroperatively linking two or more the polynucleotides encoding thefluorescent protein variants to each other.

Uses of Fluorescent Protein Variants

A fluorescent protein variant having features of the invention is usefulin any method that employs a fluorescent protein. Thus, the fluorescentprotein variants, including the monomeric, dimeric, and tandem dimerfluorescent proteins, are useful as fluorescent markers in the many waysfluorescent markers already are used, including, for example, couplingfluorescent protein variants to antibodies, polynucleotides or otherreceptors for use in detection assays such as immunoassays orhybridization assays, or to track the movement of proteins in cells. Forintracellular tracking studies, a first (or other) polynucleotideencoding the fluorescent protein variant is fused to a second (or other)polynucleotide encoding a protein of interest and the construct, ifdesired, can be inserted into an expression vector. Upon expressioninside the cell, the protein of interest can be localized based onfluorescence, without concern that localization of the protein is anartifact caused by oligomerization of the fluorescent protein componentof the fusion protein. In one embodiment of this method, two proteins ofinterest independently are fused with two fluorescent protein variantsthat have different fluorescent characteristics.

Fluorescent protein variants having features of the invention are usefulin systems to detect induction of transcription. For example, anucleotide sequence encoding a non-oligomerizing monomeric, dimeric ortandem dimeric fluorescent protein can be fused to a promoter or otherexpression control sequence of interest, which can be contained in anexpression vector, the construct can be transfected into a cell, andinduction of the promoter (or other regulatory element) can be measuredby detecting the presence or amount of fluorescence, thereby allowing ameans to observe the responsiveness of a signaling pathway from receptorto promoter.

A fluorescent protein variant of the invention also is useful inapplications involving FRET, which can detect events as a function ofthe movement of fluorescent donors and acceptors towards or away fromeach other. One or both of the donor/acceptor pair can be a fluorescentprotein variant. Such-a donor/acceptor pair provides a wide separationbetween the excitation and emission peaks of the donor, and providesgood overlap between the donor emission spectrum and the acceptorexcitation spectrum. Variant red fluorescent proteins or red-shiftedmutants as disclosed herein are specifically disclosed as the acceptorin such a pair.

FRET can be used to detect cleavage of a substrate having the donor andacceptor coupled to the substrate on opposite sides of the cleavagesite. Upon cleavage of the substrate, the donor/acceptor pair physicallyseparate, eliminating FRET. Such an assay can be performed, for example,by contacting the substrate with a sample, and determining a qualitativeor quantitative change in FRET (see, for example, U.S. Pat. No.5,741,657, which is incorporated herein by reference). A fluorescentprotein variant donor/acceptor pair also can be part of a fusion proteincoupled by a peptide having a proteolytic cleavage site (see, forexample, U.S. Pat. No. 5,981,200, which is incorporated herein byreference). FRET also can be used to detect changes in potential acrossa membrane. For example, a donor and acceptor can be placed on oppositesides of a membrane such that one translates across the membrane inresponse to a voltage change, thereby producing a measurable FRET (see,for example, U.S. Pat. No. 5,661,035, which is incorporated herein byreference).

In other embodiments, a fluorescent protein of the invention is usefulfor making fluorescent sensors for protein kinase and phosphataseactivities or indicators for, small ions and molecules such as Ca²⁺,Zn²⁺, cyclic 3′,5′-adenosine monophosphate, and cyclic 3′,5′-guanosinemonophosphate.

Fluorescence in a sample generally is measured using a fluorimeter,wherein excitation radiation from an excitation source having a firstwavelength, passes through excitation optics, which cause the excitationradiation to excite the sample. In response, a fluorescent proteinvariant in the sample emits radiation having a wavelength that isdifferent from the excitation wavelength. Collection optics then collectthe emission from the sample. The device can include a temperaturecontroller to maintain the sample at a specific temperature while it isbeing scanned, and can have a multi-axis translation stage, which movesa microtiter plate holding a plurality of samples in order to positiondifferent wells to be exposed. The multi-axis translation stage,temperature controller, auto-focusing feature, and electronicsassociated with imaging and data collection can be managed by anappropriately programmed digital computer, which also can transform thedata collected during the assay into another format for presentation.This process can be miniaturized and automated to enable screening manythousands of compounds in a high throughput format. These and othermethods of performing assays on fluorescent materials are well known inthe art (see, for example, Lakowicz, “Principles of FluorescenceSpectroscopy” (Plenum Press 1983); Herman, “Resonance energy transfermicroscopy” In “Fluorescence Microscopy of Living Cells in Culture” PartB, Meth. Cell Biol. 30:219-243 (ed. Taylor and Wang; Academic Press1989); Turro, “Modem Molecular Photochemistry” (Benjamin/Cummings Publ.Co., Inc. 1978), pp. 296-361, each of which is incorporated herein byreference).

Accordingly, the present invention provides a method for identifying thepresence of a molecule in a sample. Such a method can be performed, forexample, by linking a fluorescent protein variant of the invention tothe molecule, and detecting fluorescence due to the fluorescent proteinvariant in a sample suspected of containing the molecule. The moleculeto be detected can be a polypeptide, a polynucleotide, or any othermolecule, including, for example, an antibody, an enzyme, or a receptor,and the fluorescent protein variant can be a tandem dimer fluorescentprotein.

The sample to be examined can be any sample, including a biologicalsample, an environmental sample, or any other sample for which it isdesired to determine whether a particular molecule is present therein.Preferably, the sample includes a cell or an extract thereof. The cellcan be obtained from a vertebrate, including a mammal such as a human,or from an invertebrate, and can be a cell from a plant or an animal.The cell can be obtained from a culture of such cells, for example, acell line, or can be isolated from an organism. As such, the cell can becontained in a tissue sample, which can be obtained from an organism byany means commonly used to obtain a tissue sample, for example, bybiopsy of a human. Where the method is performed using an intact livingcell or a freshly isolated tissue or organ sample, the presence of amolecule of interest in living cells can be identified, thus providing ameans to determine, for example, the intracellular compartmentalizationof the molecule. The use of the fluorescent protein variants of theinvention for such a purpose provides a substantial advantage in thatthe likelihood of aberrant identification or localization due tooligomerization the fluorescent protein is greatly minimized.

A fluorescent protein variant can be linked to the molecule directly orindirectly, using any linkage that is stable under the conditions towhich the protein-molecule complex is to be exposed. Thus, thefluorescent protein and molecule can be linked via a chemical reactionbetween reactive groups present on the protein and molecule, or thelinkage can be mediated by linker moiety, which contains reactive groupsspecific for the fluorescent protein and the molecule. It will berecognized that the appropriate conditions for linking the fluorescentprotein variant and the molecule are selected depending, for example, onthe chemical nature of the molecule and the type of linkage desired.Where the molecule of interest is a polypeptide, a convenient means forlinking a fluorescent protein variant and the molecule is by expressingthem as a fusion protein from a recombinant nucleic acid molecule, whichcomprises a polynucleotide encoding, for example, a tandem dimerfluorescent protein operatively linked to a polynucleotide encoding thepolypeptide molecule.

A method of identifying an agent or condition that regulates theactivity of an expression control sequence also is provided. Such amethod can be performed, for example, by exposing a recombinant nucleicacid molecule, which includes a polynucleotide encoding a fluorescentprotein variant operatively linked to an expression control sequence, toan agent or condition suspected of being able to regulate expression ofa polynucleotide from the expression control sequence, and detectingfluorescence of the fluorescent protein variant due to such exposure.Such a method is useful, for example, for identifying chemical orbiological agents, including cellular proteins, that can regulateexpression from the expression control sequence, including cellularfactors involved in the tissue specific expression from the regulatoryelement. As such, the expression control sequence can be a transcriptionregulatory element such as a promoter, enhancer, silencer, intronsplicing recognition site, polyadenylation site, or the like; or atranslation regulatory element such as a ribosome binding site.

Fluorescent protein variants having features the invention also areuseful in a method of identifying a specific interaction of a firstmolecule and a second molecule. Such a method can be performed, forexample, by contacting the first molecule, which is linked to a donorfirst fluorescent protein variant, and the second molecule, which islinked to an acceptor second fluorescent protein variant, underconditions that allow a specific interaction of the first molecule andsecond molecule; exciting the donor; and detecting fluorescence orluminescence resonance energy transfer from the donor to the acceptor,thereby identifying a specific interaction of the first molecule and thesecond molecule. The conditions for such an interaction can be anyconditions under which is expected or suspected that the molecules canspecifically interact. In particular, where the molecules to be examinedare cellular molecules, the conditions generally are physiologicalconditions. As such, the method can be performed in vitro usingconditions of buffer, pH, ionic strength, and the like, that mimicphysiological conditions, or the method can be performed in a cell orusing a cell extract.

Luminescence resonance energy transfer entails energy transfer from achemiluminescent, bioluminescent, lanthanide, or transition metal donorto the red fluorescent protein moiety. The longer wavelengths ofexcitation of red fluorescent proteins permit energy transfer from agreater variety of donors and over greater distances than possible withgreen fluorescent protein variants. Also, the longer wavelengths ofemission is more efficiently detected by solid-state photodetectors andis particularly valuable for in vivo applications where red lightpenetrates tissue far better than shorter wavelengths. Chemiluminescentdonors include but are not limited to luminol derivatives andperoxyoxalate systems. Bioluminescent donors include but are not limitedto aequorin, obelin, firefly luciferase, Renilla luciferase, bacterialluciferase, and variants thereof. Lanthanide donors include but are notlimited to terbium chelates containing ultraviolet-absorbing sensitizerchromophores linked to multiple liganding groups to shield the metal ionfrom solvent water. Transition metal donors include but are not limitedto ruthenium and osmium chelates of oligopyridine ligands.Chemiluminescent and bioluminescent donors need no excitation light butare energized by addition of substrates, whereas the metal-based systemsneed excitation light but offer longer excited state lifetimes,facilitating time-gated detection to discriminate against unwantedbackground fluorescence and scattering.

The first and second molecules can be cellular proteins that are beinginvestigated to determine whether the proteins specifically interact, orto confirm such an interaction. Such first and second cellular proteinscan be the same, where they are being examined, for example, for anability to oligomerize, or they can be different where the proteins arebeing examined as specific binding partners involved, for example, in anintracellular pathway. The first and second molecules also can be apolynucleotide and a polypeptide, for example, a polynucleotide known orto be examined for transcription regulatory element activity and apolypeptide known or being tested for transcription factor activity. Forexample, the first molecule can comprise a plurality of nucleotidesequences, which can be random or can be variants of a known sequence,that are to be tested for transcription regulatory element activity, andthe second molecule can be a transcription factor, such a method beinguseful for identifying novel transcription regulatory elements havingdesirable activities.

The present invention also provides a method for determining whether asample contains an enzyme. Such a method can be performed, for example,by contacting a sample with a tandem fluorescent protein variant of theinvention; exciting the donor, and determining a fluorescence propertyin the sample, wherein the presence of an enzyme in the sample resultsin a change in the degree of fluorescence resonance energy transfer.Similarly, the present invention relates to a method for determining theactivity of an enzyme in a cell. Such a method can be performed, forexample, providing a cell that expresses a tandem fluorescent proteinvariant construct, wherein the peptide linker moiety comprises acleavage recognition amino acid sequence specific for the enzymecoupling the donor and the acceptor; exciting said donor, anddetermining the degree of fluorescence resonance energy transfer in thecell, wherein the presence of enzyme activity in the cell results in achange in the degree of fluorescence resonance energy transfer.

Also provided is a method for determining the pH of a sample. Such amethod can be performed, for example, by contacting the sample—with afirst fluorescent protein variant, which can be a tandem dimerfluorescent protein, wherein the emission intensity of the firstfluorescent protein variant changes as pH vanes between pH 5 and pH 10;exciting the indicator; and determining the intensity of light emittedby the first fluorescent protein variant. at a first wavelength, whereinthe emission intensity of the first fluorescent protein variantindicates the pH of the sample. The first fluorescent protein variantuseful in this method, or in any method of the invention, can comprisetwo DsRed monomers as set forth in SEQ ID NO: 8. It will be recognizedthat such fluorescent protein variants similarly are useful, eitheralone or in combination, for the variously disclosed methods of theinvention.

The sample used in a method for determining the pH of a sample can beany sample, including, for example, a biological tissue sample, or acell or a fraction thereof. In addition, the method can further includecontacting the sample with a second fluorescent protein variant, whereinthe emission intensity of the second fluorescent protein variant changesas pH vanes from 5 to 10, and wherein the second fluorescent proteinvariant emits at a second wavelength that is distinct from the firstwavelength; exciting the second fluorescent protein variant; determiningthe intensity of light emitted by the second fluorescent protein variantat the second wavelength; and comparing the fluorescence at the secondwavelength to the fluorescence at the first wavelength. The first (orsecond) fluorescent protein variant can include a targeting sequence,for example, a cell compartmentalization domain such a domain thattargets the fluorescent protein variant in a cell to the cytosol, theendoplasmic reticulum, the mitochondrial matrix, the chloroplast lumen,the medial trans-Golgi cisternae, a lumen of a lysosome, or a lumen ofan endosome. For example, the cell compartmentalization domain caninclude amino acid residues 1 to 81 of human type H membrane-anchoredprotein galactosyltransferase, or amino acid residues 1 to 12 of thepresequence of subunit IV of cytochrome c oxidase.

The following Examples are provided to further illustrate certainembodiments and aspects of the present invention. It is not intendedthat these Examples should limit the scope of any aspect of theinvention. Although specific reaction conditions and reagents aredescribed, it is clear that one familiar with the art would recognizealternative or equivalent conditions that also find use with theinvention, where the alternative or equivalent conditions do not departfrom the scope of the invention.

Example 1 Construction of Dimeric and Monomeric Red Fluorescent ProteinsDsRed Mutagenesis and Screening

The DsRed gene was amplified from vector pDsRed-N1 (CLONTECH, Palo Alto,Calif.) or the T1 variant (provided by B. S. Glick, University ofChicago) and subcloned into pRSET_(B) (Invitrogen™; see Baird et al.,Proc. Natl. Acad. Sci. USA 97:11984-11989 [2000]). The pRSET_(B) vectorproduces 6×His tagged fusion proteins, where an N-terminal polyhistidinetag having the following sequence is coupled to the suitably subclonedsequence:

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDP (SEQ ID NO: 22)

This resulting construct was used as the template for introduction ofthe I125R mutant using the QuikChange™ Site Directed Mutagenesis Kit(Stratagene®), according to the manufacturer's instructions. Thecomplete DsRed wild-type cDNA and polypeptide sequences are provided inGenBank Accession Number AF168419. This nucleotide sequence is alsoprovided in FIG. 16 and SEQ ID NO: 2. A variation of this nucleotidesequence is also known (Clontech), where various nucleotide positionsbeen amended to accommodate mammalian codon usage utilizationpreferences. This nucleotide sequence is provided in FIG. 25 and SEQ IDNO: 23. The corresponding polypeptide encoded by both of thesenucleotide sequences is provided in FIG. 17 and SEQ ID NO: 1.

Similarly, the DsRed T1 variant cDNA nucleotide sequence is provided inFIG. 18 and SEQ ID NO: 5. The corresponding polypeptide is provided inFIG. 19 and SEQ ID NO: 4.

As used herein, the numbering of amino acids in DsRed or mRFP1 variantsconforms to the wild-type sequence of DsRed, in which residues 66-68 ofwild-type DsRed (Gln-Tyr-Gly) are homologous to the chromophore-formingresidues 65-67 of GFP (Ser-Tyr-Gly). The amino-terminal polyhistidinetag is numbered −33 to −1. When amino acid residues are inserted at ornear position 6, they are numbered to preserve the DsRed numbering forthe rest of the protein; for example, where NNMA (SEQ ID NO:112) or DNMA(SEQ ID NO:111) are inserted at position 6, residues NNMA (or DNMA) arenumbered as residues 6a, 6b, 6c, and 6d.

Error prone PCR Mutagenesis—Error prone PCR was performed essentially asdescribed in Griesbeck et al. (J. Biol. Chem., 276:29188-29194 [2001]).Briefly, the cDNA encoding DsRed in the vector pRSET_(B) (Invitrogen™)was subjected to error-prone PCR using Taq DNA polymerase. The 5′ primerincluded a BamHI site and ended at the starting Met of the DsRed, andthe 3′ primer included an EcoRI site and ended at the stop codon,theoretically allowing mutagenesis of every base of DsRed open readingframe, except for the initiator methionine. The PCR reactions (38 cycleswith annealing at 55° C.) were run in four 100 μL batches, eachcontaining 10 μL of 10×PCR buffer with Mg²⁺ (Roche MolecularBiochemicals), 150 μM Mn²⁺, 250 μM of three nucleotides, 50 μM of theremaining nucleotide, and 5 ng of template DNA.

Mutagenized PCR products were combined, purified by agarose gelelectrophoresis, digested with BamHI and EcoRI, and isolated by QIAGEN®QIAquick™ DNA purification spin column following the manufacturer'sinstructions. The resulting fragments were ligated into pRSET_(B), andthe crude ligation mixture was transformed into E. coli BL21(DE3) Gold(Stratagene®) by electroporation.

Overlap Extension PCR Mutagenesis—Semi-random mutations at multipledistant locations were introduced by overlap extension PCR with multiplefragments essentially as described in Ho et al., Gene 77:51-59 (1989).Briefly, two to four pairs of sense and antisense oligonucleotideprimers (Invitrogen™ or GenBase), with semi-degenerate codons atpositions of interest, were used for PCR amplification of the DsRedtemplate with Pfu DNA polymerase (Stratagene®) in individual reactions.The resulting overlapping fragments were gel purified using QIAGEN® gelextraction kit and recombined by overlap extension PCR with Pfu or TaqDNA polymerase (Roche).

Full length genes were digested with BamHI/EcoRI (New England BioLabs®)and ligated into pRSET_(B) with T4 ligase (New England BioLabs®).Chemically competent E. coli JM109(DE3) were transformed and grown onLB/agar at 37° C.

Bacterial Fluorescence Screening—Bacteria plated on LB/agar plates werescreened essentially as described in Baird et al., Proc. Natl. Acad.Sci. USA 96:11241-11246 (1999). Briefly, the bacterial plates wereilluminated with a 150-W Xe lamp using 470 nm (40 nm bandwidth), 540 nm(30 nm bandwidth), or 560 nm (40 nm bandwidth) excitation filters and530 nm (40 nm bandwidth), 575 nm (long pass), or 610 nm (long pass)emission filters. Fluorescence was imaged by a cooled charge-coupleddevice camera (Sensys Photometrics, Tucson, Ariz.) and were processedusing Metamorph software (Universal Imaging, West Chester, Pa.).

Fluorescent colonies of interest were cultured overnight in 2 ml of LBsupplemented with ampicillin. Bacteria were pelleted by centrifugationand imaged again to ensure that the protein was expressed well inculture. For fast maturing proteins a fraction of the cell pellet wasextracted with B-per II (Pierce) and complete spectra obtained. DNA waspurified from the remaining pellet by QIAGEN® QIAprep® plasmid isolationspin column according to the manufacturer's instructions and submittedfor DNA sequencing. To determine the oligomeric state of DsRed mutants,a single colony of E. coli was restreaked on LB/agar and allowed tomature at room temperature. After 2 days to 2 weeks the bacteria werescraped from the plate, extracted with B-per II, analyzed (not boiled)by SDS-PAGE (BioRad), and the gel imaged with a digital camera.

Bacterial Transformations and DsRed Protein Purification

Ligation mixtures were transformed into Escherichia coli BL21(DE3) Gold(Stratagene) by electroporation in 10% glycerol with a ligation mixture(0.1 cm cuvette, 12.5 kV/cm, 200Ω, 25 μF).

Protein was expressed and purified essentially as-described in Baird etal., Proc. Natl. Acad. Sci. USA 96:11241-11246 (1999). Briefly, whencultured for protein expression, transformed bacteria were grown to anOD₆₀₀ of 0.6 in LB containing 100 mg/liter ampicillin, at which timethey were induced with 1 mM isopropyl β-D-thiogalactoside. Bacteria wereallowed to express recombinant protein for 6 hr at room temperature andthen overnight at 4° C. The bacteria then were pelleted bycentrifugation, resuspended in 50 mM Tris-HCl/300 mM NaCl, and lysed bya French press. The bacterial lysates were centrifuged at 30,000×g for30 min, and the proteins were purified from the supernatants usingNi-NTA resin (QIAGEN®).

Spectroscopy of purified protein was typically performed in 100 mM KCl,10 mM MOPS, pH 7.25, in a fluorescence spectrometer (Fluorolog-2, SpexIndustries). All DNA sequencing was performed by the Molecular PathologyShared Resource, University of California, San Diego, Cancer Center.

Construction of DsRed Tandem Dimers and Constructs for Mammalian CellExpression, Including Chimeric Constructs

To construct tandem dimers of DsRed protein, dimer2 in pRSET_(B) wasamplified in two separate PCR reactions. In the first reaction, the 5′BamHI and a 3′ SphI site were introduced while in the second reaction a5′ SacI and a 3′ EcoRI site were introduced. The construct was assembledin a 4-part ligation containing the digested dimer2 genes, a syntheticlinker with phosphorylated sticky ends, and digested pRSET_(B). Fourdifferent linkers were used, which encoded polypeptides of variouslengths. These were:

SEQ Linker Polypeptide Sequence ID NO  9 a.a. residue linker RMGTGSGQL16 12 a.a. residue linker GHGTGSTGSGSS 17 13 a.a. residue linkerRMGSTSGSTKGQL 18 22 a.a. residue linker RMGSTSGSGKPGSGEGSTKG 19 L(“a.a.” indicates amino acid)

For expression in mammalian cells, DsRed variants were amplified frompRSET_(B) with a 5′ primer that encoded a KpnI restriction site and aKozak sequence. The PCR product was digested, ligated into pcDNA3, andused to transform E. coli DH5α.

A gene encoding a chimeric fusion polypeptide comprising DsRed andconnexin43 (Cx43) was constructed. To produce these fusions, Cx43 wasfirst amplified with a T primer encoding a seven-residue linker endingin a BamHI site. The construct was assembled in a 3-part ligationcontaining KpnI/BamHI digested Cx43, BamHI/EcoRI digested enhanced GFP,and digested pcDNA3. For all other fusion proteins (Cx43-T1, -dimer2,-tdimer2(12) and -mRFP1) the gene for the fluorescent protein wasligated into the BamHI/EcoRI digested Cx43-GFP vector.

DsRed Protein Variant Production and Characterization

DsRed variants were expressed essentially as described in Baird et al.,Proc. Natl. Acad. Sci. USA 96:11241-11246 (1999). All proteins werepurified by Ni-NTA chromatography (QIAGEN®) according to themanufacturer's instructions and dialyzed into 10 mM Tris, pH 7.5 orphosphate buffered saline supplemented with 1 mM EDTA. All biochemicalcharacterization experiments were performed essentially as described inBaird et al., Proc. Natl. Acad. Sci. USA 97:11984-11989 (2000).

The maturation time courses were determined on a Safire 96 well platereader with monochromators (TECAN, Austria). Aqueous droplets ofpurified, protein in phosphate-buffered saline were formed under mineraloil in a chamber on the fluorescence microscope stage. For reproducibleresults it proved essential to pre-extract the oil with aqueous buffer,which would remove any traces of autoxidized or acidic contaminants. Thedroplets were small enough (5-10 μm diameter) so that all the moleculeswould see the same incident intensity. The absolute excitationirradiance in photons/(cm²·s·nm) as a function of wavelength wascomputed from the spectra of a xenon lamp, the transmission of theexcitation filter, the reflectance of the dichroic mirror, themanufacturer-supplied absolute spectral sensitivity of a miniatureintegrating-sphere detector (SPD024 head and ILC1700 meter,International Light Corp., Newburyport, Mass.), and the measureddetector current. The predicted rate of initial photon emission (beforeany photobleaching had occurred) was calculated from the excitationirradiance and absorbance spectrum (both as functions of wavelength),and the quantum yield. These rates varied. from 180s⁻¹ for mHoneydew to3300s⁻¹ for mStrawberry. To normalize the observed photobleaching timecourses to a common arbitrary standard of 1000 emitted photons/sec, thetime axes were correspondingly scaled by factors of 0.18 to 3.3,assuming that emission and photobleach rates are both proportional toexcitation intensity at intensities typical of microscopes with arc lampsources, as is known to be the case for GFP.

Analytical Ultracentrifugation—Purified, recombinant DsRed was dialyzedextensively against PBS, pH 7.4 or 10 mM Tris, 1 mM EDTA, pH 7.5.Sedimentation equilibrium experiments were performed on a Beckman OptimaXL-I analytical ultracentrifuge at 20° C. measuring absorbance at 558 nmas a function of radius. Samples of DsRed were normalized to 3.57 μM(0.25 absorbance units), and from this, 125 μL aliquots were loaded intosix channel cells. The data were analyzed globally at 10K, 14K, and 20Krpm by nonlinear least-squares analysis using the ORIGIN softwarepackage supplied by Beckman. The goodness of fit was evaluated on thebasis of the magnitude and randomness of the residuals, expressed as thedifference between the experimental data and the theoretical curve andalso by checking each of the fit parameters for physical reasonability.

Absorption/Fluorescence Spectra and Extinction Coefficients—Fluorescencespectra were taken with a Fluorolog spectrofluorimeter (Spex Industries,Edison, N.J.). Absorbance spectra of proteins were taken with a CaryITV-V is spectrophotometer. For quantum yield determination, thefluorescence of a solution of DsRed or DsRed variant in PBS was comparedwith equally absorbing solutions of rhodamine B and rhodamine 10 l inethanol. Corrections were included in the quantum yield calculation forthe refractive index difference between ethanol and water. Forextinction coefficient determination, native protein absorbance wasmeasured with the spectrophotometer, and protein concentration wasmeasured by the BCA method (Pierce).

Mammalian Cell Imaging and Microinjection

HeLa cells were transfected with DsRed variants or Cx43-DsRed fusions inpcDNA3 through the use of Fugene 6 transfection reagent (Roche).Transfected cells were grown for 12 hours to 2 days in DMEM at 37° C.before imaging using a Zeiss Axiovert 35 fluorescence microscope withcells in glucose-supplemented HBSS at room temperature. Individual cellsexpressing Cx43 fused to a DsRed variant, or contacting non-transfectedcells for control experiments, were microinjected with a 2.5% solutionof lucifer yellow (Molecular Probes, Eugene, Oreg.). Images wereacquired and processed with the Metafluor software package (UniversalImaging, West Chester, Pa.).

Results

Stepwise Evolution of DsRed Molecules

The present invention provides methods for the stepwise evolution oftetrameric DsRed to a dimer and then either to a genetic fusion of twocopies of the protein, i.e., a tandem dimer, or to a true monomerdesignated mRFP1. Each subunit interface was disrupted by insertion ofarginines, which initially crippled the resulting protein, but redfluorescence could be rescued by random and directed mutagenesistotaling 17 substitutions in the dimer and 33 substitutions in mRFP1Fusions of the gap junction protein.connexin43 to mRFP1 formed fullyfunctional junctions, whereas analogous fusions to the tetramer anddimer failed. Although mRFP1 has somewhat lower extinction coefficient,quantum yield, and photostability than DsRed, mRFP1 matures >10× faster,so that it shows similar brightness in living cells. In addition, theexcitation and emission peaks of mRFP1, 584 and 607 nm, are ˜25 nm redshifted from DsRed, which should confer greater tissue penetration andspectral separation from autofluorescence and other fluorescentproteins.

The consensus view is that a monomeric form of DsRed will be essentialif it is to ever reach its full potential as a genetically encoded redfluorescent tag (Remington, Nat. Biotechnol., 20:28-29 [2002]). Thepresent invention provides a directed evolution and preliminarycharacterization of the first monomeric red fluorescent protein. Thepresent invention provides an independent alternative to GFP in theconstruction of fluorescently tagged fusion proteins.

Directed and Random Evolution of a Dimer of DsRed

The basic strategy for decreasing the oligomeric state of DsRed was toreplace key hydrophobic residues at the dimer interface by chargedresidues such as arginine. The high energetic cost of burying a chargedresidue within a nonpolar hydrophobic interface or of placing twopositive charges in close proximity should disrupt the interaction.Initial attempts to break apart the DsRed AC interface (see FIG. 2A)with the single mutations T147R, H162R, and F224R, consistently gavenon-fluorescent proteins. The AB interface however, proved somewhat lessresilient and could be broken with the single mutation I125R to give apoorly red fluorescent dimer that suffered from an increased greencomponent and required more than 10 days to fully mature.

To reconstitute the red fluorescence of DsRed-I125R, the protein wassubjected to iterative cycles of evolution. This accelerated evolutionstrategy used either random mutagenesis or semi-directed mutagenesis tocreate a library of mutated molecules, which can be screened fordesirable characteristics. The directed evolution strategy of thepresent invention is shown in FIG. 7. Each cycle of the mutagenesisbegan with random mutagenesis to identify those positions that effectedeither the maturation or brightness of the red fluorescent protein. Onceseveral residues were identified, expanded libraries were constructed inwhich several of these key positions were simultaneously mutated to anumber of substitutions (see FIGS. 10-12). These directed librariescombine the benefits of shuffling of improved mutant genes with anefficient method of overcoming the limited number of substitutionsaccessible during random mutagenesis by error prone PCR. Most methods ofin vitro recombination rely on random gene fragmentation. In contrast,the methods of the present invention use PCR to generate designedfragments that can be reassembled to give the full length shuffled gene.

Libraries of mutant red fluorescent proteins were screened in coloniesof E. coli and were evaluated on both the magnitude of their redfluorescence under direct excitation at 540 nm and the ratio of emissionintensities at 540 nm over 470 nm excitation. While the formerconstraint selected for very bright or fast maturing mutants, the latterconstraint selected for mutants with decreased 470 nm excitation orred-shifted excitation spectra. Multiple cycles of random mutagenesiswere used to find sequence locations that affected the maturation andbrightness of the protein, and then expanded libraries of mutations atthose positions were created and recombined to find optimalpermutations.

Initial random mutagenesis of DsRed-I125R identified several beneficialmutations including K163Q or M, S179T and T217S. These three positionswere included in our first directed library in which a total of sevenresidues were simultaneously mutated to a number of reasonablesubstitutions. The additional positions targeted in the first directedlibrary included N42 and V44, residues that are critical for the fastphenotype of T1 (Bevis and Glick, Nat. Biotechnol., 20:83-87 [2002]).Also included were I161 and S197, positions at which specific mutationscontributed to the modest improvements of DSRed2 (CLONTECH) and the verysimilar ‘E57’ (Terskikh et al., J. Biol. Chem., 277:7633-7636 [2002]).From this library, several clones were identified such as DsRed-I125R,S179T, T217A and DsRed-I125R, K163Q, T217A, but improvements were notdramatic.

As an alternative strategy, the DsRed variant fast tetramer T1 (Bevisand Glick, Nat. Biotechnol., 20:83-87 [2002]) was also studied.Introduction of the I125R mutation into this protein (T1 DsRed-I125Rpolypeptide sequence provided in SEQ ID NO: 24) resulted in a dimer thatmatured in only a few days, which was comparable to the best DsReddimers produced at that time. By further targeting those positions thathad helped rescue DsRed-I125R, dramatic improvements in our firstgeneration library were observed.

A similar directed mutagenesis strategy starting from T1-I125R (see FIG.10A, library D1) was undertaken and eventually identified dimer1. Dimer1was somewhat better than wt DsRed both in terms of brightness and rateof maturation but had a substantial green peak equivalent to that of T1.Dimer1 was also somewhat blue-shifted with an excitation maximum at 551nm and an emission maximum at 579 nm. Error prone PCR on Dimer1 (FIG.10A, library D2) resulted in the discovery of Dimer1.02 containing themutation V71A in the hydrophobic core of the protein and effectively nogreen component in the excitation spectra. A second round of randommutagenesis (FIG. 10A, library D3) identified the mutations K70R whichfurther decreased the green excitation, S197A which red-shifted thedimer back to DsRed wavelengths and T217S which greatly improved therate of maturation. Unfortunately, K70R and S197A matured relativelyslowly and T217S had a green excitation peak equivalent to DsRed. Usingdimer1.02 as the template, two more rounds of directed mutagenesis wereperformed; the first focusing on the three positions identified above(FIG. 11A, library D3) and the second on C117, F118, F124, and V127(FIG. 10A, library D4).

Continuing with the directed evolution strategy for a total of 4generations, an optimal dimeric variant was produced, which wasdesignated dimer2 (illustrated in FIG. 2B). This variant contains 17mutations, of which eight are internal to the β-barrel (N42Q, V44A,V71A, F118L, K163Q, S179T, S197T and T217S), three are the aggregationreducing mutations found in T1 (R2A, K5E and N61) and see Bevis andGlick, Nat. Biotechnol., 20:83-87 [2002]; and Yanushevich et al., FEBSLett., 511:11-14 [2002]), two are AB interface mutations (I125R andV127T), and 4 are miscellaneous surface mutations (T21S, H41T, C117T andS131P). The dimer2 nucleotide sequence is provided in SEQ ID NO: 7 andFIG. 21. The dimer2 polypeptide is provided in SEQ ID NO: 6 and FIG.22A.

A variant of dimer2 (SEQ ID NO:6) may comprise one or more amino acidsubstitutions selected from amino acid substitutions at positions 22,66, 105, and 124, and may also include terminal amino acids at the GFPterminus. A variant of dimer2 (SEQ ID NO:6) may have about 80%, or 85%,or 90%, or 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity with the amino acid sequence of SEQ ID NO:6. For example,substitutions in the dimeric protein may be selected from one or more ofV22M, Q66M, V104L, and F124M. In a preferred embodiment, the proteinvariant is dimer2.2MMM (dimer3) (dTomato) (SEQ ID NO: 81) havingsubstitutions V22M, Q66M, V104L, and F124M with respect to SEQ ID NO: 6and having GFP termini of MVSKGEE (SEQ ID NO: 14) (at the N-terminus)and GMDELYK (SEQ ID NO: 91) or YGMDELYK (SEQ ID NO: 110) at theC-terminus. The underlined residues were copied from the N- andC-termini of EGFP (containing mutations F64L, S65T; SEQ ID NO: 13) andreplace the corresponding amino acids in DsRed and mRFP1, as illustratedin FIG. 22B. Also shown in FIG. 22B are the N-terminal amino acidsequences of DsRed (SEQ ID NO: 1) and mRFP1 (SEQ ID NO: 8).

As shown in the table of FIG. 32, dimer2.2MMM (dimer3) (dTomato) has anexcitation wavelength peak at 554 nm and an emission wavelength peak at581 nm, with an extinction coefficient of 70,000 M⁻¹cm⁻¹ and a quantumyield of 0.72. Dimer2.2NIMM (dimer3) (dTomato) has the highestextinction coefficient of the variants that have been made, and hasimproved quantum yield as compared to dimer2. Its excitation andemission wavelengths are most similar to the wild type DsRed, with hasvery little green component (having less green component than either wtDsRed or dimer2).

Construction of a Tandem Dimer of DsRed

In an attempt to produce a still further advantageous form of DsRed, analternative novel strategy to synthesize a more stable DsRed dimer wasdevised. This approach utilized covalent tethering of two engineeredmonomeric DsRed units to yield a dimeric form of DsRed with advantageousproperties. The basic strategy was to fuse two copies of an AC dimerwith a polypeptide linker such that the critical dimer interactionscould be satisfied through intramolecular contacts with the tandempartner encoded within the same polypeptide.

Based on the crystal structure of the DsRed tetramer (Yarbrough et al.,Proc. Natl. Acad. Sci. USA 98:462-467 [2001]; and Wall et al., NatureStruct. Biol., 7:1133-1138 [2000]), it was contemplated that a 10 to 20residue linker could extend from the C-terminus of the A subunit to theN-terminus of the C subunit (˜30 Å, see FIG. 1B), but not to theN-terminus of the B subunit (>70 Å). Using the optimized dimer2, aseries of four tandem constructs were produced using linkers of varyinglengths (9, 12, 13, or 22 amino acids) comprising a sequence similar toa known protease resistant linker (Whitlow et al. Protein Eng.,6:989-995 [1993]).

Of the four constructions, only the tandem construct with the 9 residuelinker was notable for a somewhat slower maturation. The other threeconstructs were practically indistinguishable in this respect, and thus,find equal use with the present invention. The tandem dimer constructwith the 12 residue linker, designated tdimer2(12), was used in allsubsequent experiments. As expected, dimer2 and tdimer2(12) haveidentical excitation and emission maximum and quantum yields (see FIG.14). However, the extinction coefficient of tdimer2(12) is twice that ofdimer2 due to the presence of two equally absorbing chromophores perpolypeptide chain.

Evolution of a Monomeric DsRed

In an attempt to create improved dimers of DsRed would better toleratedisruption of the remaining interface, libraries were constructed whereAC interface breaking mutations were incorporated into the tdimer2(12):An initial dimer library was reassembled using a 3′ primer that encodedthe mutations H222G and F224G (FIG. 10A, library D5). These two residuesform the bulk of the dimer contacts in the C-terminal tail of DsRed thathooks around the C-terminal tail of the dimer partner. From this librarythe best two unique clones, HF2Ga and HF2Gb, were very similar insequence to Dimer1 with the primary differences being the mutationsF124L present in both clones, K163H in HF2 Gb and the H222G and F224Greplacements. Both HF2Ga and HF2 Gb migrated as fluorescent dimers whenloaded unboiled onto a 12% SDS-PAGE gel so they must maintain a stabledimer interface.

Simultaneously, a more direct approach to breaking up the AC interfacethrough introduction of dimer-breaking mutations was undertaken. Dimer1was the template for the first such library (FIG. 10A, library M1) inwhich nine different positions were targeted, including two key ACinterface residues, H162 and A164, which were substituted for lysine orarginine, respectively. The brightest colonies from this library weredifficult to distinguish from the background red fluorescence of the E.coli colonies even after prolonged imaging with a digital camera.Suspect colonies were restreaked on LB/agar, allowed to mature at roomtemperature for two weeks and a crude protein preparation analyzed bySDS-PAGE. Imaging of the gel revealed a single faint band consistentwith the expected mass of the monomer. Thus, this species was termedmRFP0.1 (for monomeric Red Fluorescent Protein). Sequencing of thisclone revealed that mRFP0.1 was equivalent to Dimer1 with mutationsE144A, A145R, H162K, K163M, A164R, H222G and H224G.

Random mutagenesis on mRFP0.1 (FIG. 10A, library M2) resulted in thecreation of the much brighter mRFP0.2, which gave an unambiguous redfluorescent and monomeric band by SDS-PAGE, and which contained thesingle additional mutation Y192C. Both mRFP0.1 and mRFP0.2 displayed atleast 3-fold greater green fluorescence than red fluorescence, but asexpected for the monomer, there was no FRET between the green and redcomponents.

With the suspicion that mutations that were beneficial to the dimercould also benefit the monomer, a template mixture including mRFP0.2,Dimer1.56, HF2Ga and HG2Gb was subjected to a combination of PCR-basedtemplate shuffling and directed mutagenesis (FIG. 10A, library M3). Thetop clone identified in this library, mRFP0.3 was relatively bright andhad a greatly diminished green fluorescent component. In addition,mRFP0.3 was approximately 10 nm red shifted from DsRed and was primarilyderived from Dimer1.56.

The goal of the next directed library (FIG. 10B, library M4) was toinvestigate the effect of mutations at K83, which have previously beenshown to cause a red shift in DsRed (Wall et al., Nature Struct. Biol.,7:1133-1138 [2000]). The top two clones, designated mRFP0.4a andmRFP0.4b, contained the K83I or L mutation respectively, were 25 nm redshifted relative to DsRed and were very similar in terms of maturationrate and brightness. Unlike all the previous generations of the monomer,colonies of E. coli transformed with mRFP0.4a were red fluorescentwithin 12 h after transformation when excited with 540 nm light andviewed through a red filter.

A template mixture of mRFP0.4a and mRFP0.4b was subjected to randommutagenesis (FIG. 10B, library M5) and the resulting library wasthoroughly screened. The 5 fastest maturing clones from this librarywere derived from mRFP0.4a and contained individual mutations L174P,V175A (two clones), F177C and F177S. The F177S clone or mRFP0.5a,appeared to mature slightly faster and had the smallest green peak inthe absorbance spectra. One colony isolated from this library wasexceptionally bright when grown on LB/agar but expressed very poorlywhen grown in liquid culture. This clone, designated mRFP0.5b, wasderived from mRFP0.4b and contained two new mutations; L150M inside thebarrel and V156A outside.

The next library (FIG. 10B, library M6) was intended to optimize theregion around residues V175 and F177 in both mRFP0.5a and theincreasingly divergent mRFP0.5b. The top clone in this library,designated mRFP0.6, was derived from mRFP0.5b, though of three other topclones, one was derived from mRFP0.5b; one from mRFP0.5a, and oneappeared to have resulted from multiple crossovers between the twotemplates—The final library (FIG. 10B, library M7) targeted residues inthe vicinity of L150 because this was the one remaining criticalmutation that was derived from random mutagenesis and had not beenreoptimized. Top clones had combinations of mutations at all targetedpositions though the clone with the single mutation R153E was found toexpress slightly better in culture. This clone was further modifiedthrough deletion of the unnecessary V1a insertion and replacement of thecysteine at position 222 with a serine.

The final clone in this series, designated mRFP1, contained a total of33 mutations (see FIG. 1C) relative to wild-type DsRed. Of thesemutations, 13 are internal to the β-barrel (N42Q, V44A, V71A, K83L,F124L, L150M, K163M, V175A, F177V, S179T, V 195T, S1971 and T217A). Ofthe 20 remaining external mutations, three are the aggregation reducingmutations from T1 (R2A, K5E and N61)), three are AB interface mutations(I125R, V127T and I180T), ten are AC interface mutations (R153E, H162K,A164R, L174D, Y192A, Y194K, H222S, L223T, F224G and L225A), and fouradditional mutations (T21S, H41T, C117E and V156A). The mRFP1 nucleotideand polypeptide sequences are provided in SEQ ID NOS: 9 and 8,respectively.

In other embodiments, the monomeric variant is a variant of mRFP1 (SEQID NO: 8). Based on indications that substitution of methionine at thefirst position in the chromophore had beneficial effects on maturationof the I125R dimeric variant of DsRed (Baird, G. S. (2001) Ph.D. thesis,University of California, San Diego), a directed library of residuesnear the chromophore was constructed, including randomization atposition 66 in mRFP1. The Q66M substitution was found to have a largeimpact on the amount of protein that assumed the mature chromophoreconformation relative to mRFP1, and the additional mutation T147S,present due to a PCR error in the Q66M mutant, was also found to bebeneficial. In addition to allowing more complete maturation, the Q66Mmutation also provides an additional red-shift of the excitation andemission spectra of approximately 5 nm relative to mRFP1. The Q66M,T147S mutant was designated mRFP1.1.

Because mRFP1 was found to be somewhat sensitive to N-terminal fusions,it was reasoned that changes to the N- and C-termini could potentiallybe beneficial to the protein in the context of fusions to otherproteins. Thus, the first and last seven amino acids of mRFP 1.1 werereplaced with the corresponding residues from GFP, with the hope thatthese residues would act as an “insulator” against adverse effects dueto fusions to the N- or C-terminus of the resulting protein. This mutantwas designated mRFP 1.2, and was found to be less sensitive to thepresence of an N-terminal 6×His tag than its predecessor. An additionalbenefit of these changes to the N- and & termini of mRFP is that primersused to amplify GFP-based proteins can now also be used for mRFP-basedproteins as well, simplifying subcloning procedures.

While the substitution of the first and last seven amino acids of mRFPfor the corresponding GFP residues was found to be beneficial, furtherreduction of the sensitivity of mRFP to fusions to its N-terminus wasdesirable. A library based on mRFP 1.2 was next constructed in whichfour additional randomized codons were inserted after position 6 in aconstruct that included an N-terminal 6×His tag. From this library, itwas found that the insertion of the amino acid sequence NNMA gave thestrongest fluorescence signal in bacteria, and so this clone wasdesignated mRFP1.3.

Additional random libraries based on mRFP1.3, as well as error-pronelibraries of wavelength-shifted mRFP variants, were constructed. Theselibraries provided data that identified the V7I and M182K mutations asbeneficial to efficient protein folding, and so these two mutations wereadded to mRFP1.3 to give mRFP1.4. It is worth noting that in homologousfluorescent proteins, position 182 is invariably occupied by apositively charged residue.

The chromophore-interacting residue 163 had not been re-investigatedsince early in the evolution of mRFP. Randomization of residue 163showed that the substitution M163Q resulted in a nearly completedisappearance of the absorbance peak at ˜510 nm present in all previousmRFP clones. This mutation additionally blue-shifted the excitation peakapproximately 2 nm relative to mRFP 1.4 while having little impact onthe emission peak of the protein, and so effectively increased theStokes shift of this mRFP variant relative to previous clones. Whilethis clone suffered from a reduced extinction coefficient, suggestingthat it too did not fold efficiently, the additional mutation R17Hrestored an extinction coefficient nearly equivalent to mRFP 1.4, givingthe variant mRFP 1.5, which is red-shifted 5 nm from mRFP1 and exhibitsnearly complete maturation with kinetics as fast or faster than mRFP1.

An additional directed library of mRFP 1.5 was constructed thatpartially randomized positions 194, 195, 196, 197, and 199, based onevidence from wavelength-shifted mRFP variants that this region couldinfluence solvent accessibility of the chromophore. From this library,the clone containing the mutations K194N, T195V, and D 196N was found tohave an enhanced extinction coefficient, while retaining the nearlycomplete lack of a 510 nm absorbance peak. This clone was designatedmRFP1.5.4 (also termed mRFP2, or mCherry).

Dimer3

The dimer2 variant previously described possesses quite desirableproperties, such as much faster maturation than wild-type DsRed andnearly complete maturation, as well as a high extinction coefficient andquantum yield relative to the fast-maturing mutant T1. In order toimprove these properties, randomly mutagenized libraries of dimer2 wereconstructed. The V 104L mutation was identified as improving thebrightness of the dimer, and subsequent analysis showed that the effectof this mutation was to significantly increase the quantum yield of theprotein to a level comparable to wild-type DsRed. However, it wasdetermined that this mutation alone was not clearly beneficial, as italso increased the proportion of protein trapped in an immature, greenfluorescent state.

As with mRFP, a decrease in the dimer RFP's sensitivity to N- andC-terminal fusions was desired, and so, as with mRFP, its first and lastseven amino acids were replaced with the corresponding GFP residues. Theresulting protein was found to have more consistent brightnessregardless of the N-terminal sequence attached to it. The V104L dimervariant with the GFP-type termini was designated dimer2.2.

Additional rounds of error-prone mutagenesis identified positions 22 and124, both of which interact with the chromophore-containing centralalpha-helix, to be important in improving the brightness of the dimer.Therefore, directed libraries were constructed at both of thesepositions; these libraries identified V22M and F124M as the optimalmutations. In addition, because the Q66M mutation had been shown toimprove the properties of both wild-type DsRed and mRFP, this mutationwas included in the resulting dimer, giving a protein differing from theoriginal dimer2 by the mutations V22M, Q66M, V104L, and F124M, as wellas the substitution of its first and last seven amino acids with thecorresponding GFP residues. This final variant was designated dimer3(dTomato), and has an improved extinction coefficient and quantum yieldrelative to dimer2, and also has improved maturation kinetics, leadingto a decreased green fluorescent component relative to dimer2.

Wavelength-Shifted Variants of mRFP

Rather than attempting to monomerize novel fluorescent proteins, it wasreasoned that it should be possible to alter the excitation and emissionwavelength of mRFP by rational mutagenesis, as had been done withAequorea GFP. Following the example of GFP, substitutions at the firstchromophore residue, Q66, and in the stacking position homologous toT203 in GFP, mRFP 1197 (S197 in wild-type DsRed) were explored.

The most significant wavelength shift we observed occurred when Q66 wassubstituted with either serine, threonine, or cysteine. All threesubstitutions gave a similar blue-shift in both excitation and emissionwavelength, and led to a curious increase in pH sensitivity,characterized by an additional blue-shift in excitation and emission atalkaline pH. Such pH-sensitivity was obviously undesirable, and sincethe additionally blue-shifted chromophore species at high pH appeared tohave increased extinction coefficient and quantum yield, we sought firstto find mutants that favored this form of the chromophore and thus hadreduced pH sensitivity.

Randomly mutagenized libraries of Q66C/S/T libraries identified theT1951 mutation as important in reducing pH sensitivity and favoring themore blue-shifted form of the mOFP chromophore. Additional directedlibraries at this and surrounding positions identified T195V as theoptimal mutation for reducing pH sensitivity, and Q66T as having thehighest quantum yield of the mOFP chromophores. This clone wasdesignated mOFP.T.12, and, in addition to the modified N- and C-terminipresent in mRFP 1.4, has the additional mutations Q66T, K194M, T195V,D196S relative to mRFP1.4.

Randomization of the stacking position, 1197, in already blue-shiftedmRFP variants led to the surprising discovery that a glutamate in thisposition provided an additional red-shift, yielding an mRFP variant withexcitation and emission spectra unlike anything yet found in nature.This true yellow mRFP variant, designated mYOFP for yellow-orangefluorescent protein, was found initially to have extreme pH sensitivityas well as inefficient maturation: Additional mutations were discoveredthrough several rounds of random and directed libraries that improvedthe folding efficiency and pH sensitivity of mYOFP variants, yieldingthe brightly fluorescent variant mYOFP 1.3, which, in addition to themodified N- and C-termini present in mRFP1.3, contains the mutationsQ66C, A77T, D78G, L83F, T108A, T147S, D174S, V177T, M182K, K1941, T195A,D196G, I197E, L1991, and Q213L relative to mRFP1.3. Interestingly, inthe case of mYOFP, the L1991 mutation had the greatest effect on pHsensitivity, while in the case of mOFP, the T197V mutation had a similareffect, while mutations at L199 have little effect.

In order to mimic the red-shift of YFPs relative to GFP, aromaticresidues were placed in the stacking position of mRFP and selected forvariants with red-shifted excitation and emission spectra. Followingseveral rounds of random and directed mutagenesis, we identified aclone, mFRFP.F2Q6, which has excitation and emission peaks ˜20 nmred-shifted relative to mRFP2, or ˜25 nm red-shifted relative to mRFP1.Significantly, this protein has an excitation maximum of 605 nm, whichis beyond the generally agreed-upon barrier of hemoglobin and cytochromeabsorbance in biological tissues. In addition to the modified N- andC-termini present in mRFP1.3, this variant contains the mutations V7I,E32K, A77P, L83M, R125H, T147S, M150L, I161V, T195L, and 1197Y relativeto mRFP1.3.

The properties of several variants of mRFP1 (SEQ ID NO:8) are presentedin the table in FIG. 32. Nucleotide sequences coding for these variantsare presented in FIGS. 34-37. A variant of mRFP1 (SEQ ID NO:8) may haveabout 80%, or 85%, or 90%, or 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity with the amino acid sequence of SEQ ID NO:8. Asindicated in FIG. 32, a variant of mRFP1 (SEQ ID NO:8) may comprise morethan one amino acid substitution as compared to the wtDsRed sequence(SEQ ID NO: 1) or mRFP1 (SEQ ID NO: 8) and may also include terminalamino acid additions or substitutions selected from the insertionscomprising one or more amino acids homologous to the amino acids at theGFP terminus, the amino acids DNMA, and the amino acids NNMA. Amino acidsubstitutions found in the protein variants dimer 2.2MMM (dimer 3)(dTomato), mRFP1.5, OrS4-9, Y1.3 (mYOFP1.3) (mBanana), and mFRFP (F2Q6)(mGrape2) are indicated in FIG. 32, which also includesexcitation/emission wavelength values, extinction coefficient values,quantum yield values, and comments on the properties of the variants.

Characterization of dimer2, tdimer2(12) and mRFP1

Initial evidence for the monomeric structure of mRFP1 and its precursorswas based on SDS-PAGE results (see FIG. 8) and the lack of FRET betweenthe green and red fluorescent components in early generations. Thusanalytical equilibrium ultracentrifugation was performed on DsRed,dimer2, and mRFP0.5a (an evolutionary precursor to mRFP1). The mRFP0.5apolypeptide sequence is illustrated in FIGS. 20A-20D. The analyticalequilibrium analysis confirmed the expected tetramer, dimer, and monomerconfigurations of the tested species (see FIGS. 3A-3C).

In a fluorescence and absorption spectra analysis, DsRed, T1 and dimer2all have a fluorescent component that contributes at 475-486 nm to theexcitation spectra due to FRET between oligomeric partners (see FIGS.4A-4C). In this analysis, the T1 peak is quite pronounced (FIG. 4B), butin dimer2 (FIG. 4C), any excitation shoulder near 480 nm is almostobscured by the 5 nm blue-shifted excitation peak. The 25 nm red-shiftedmonomeric mRFP1 (FIG. 4D) also has a peak at 503 nm in the absorptionspectra, but in contrast to the other variants, this species isnon-fluorescent mutant and therefore does not show up in the excitationspectrum collected at any emission wavelength. When the 503 nm absorbingspecies is directly excited, negligible fluorescence emission isobserved at any wavelength.

As shown in FIG. 5, the rate of maturation of dimer2, tdimer2(12) andmRFP1 is greatly accelerated over that of DsRed; though only mRFP 1matures at least as quickly as T1. Based on data collected at 37° C.,the t_(0.5) for maturation of mRFP1 and T1 are less than 1 hour. E. colicolonies expressing either dimer2 or mRFP1 display similar or brighterlevels of fluorescence to those expressing T1 after overnight incubationat 37° C. (see FIG. 9).

Expression of dimer2, tdimer2(12) and mRFP1 in Mammalian Cells

The fluorescence of the dimer2, tdimer2(12) and mRFP1 proteins in thecontext of mammalian cells was tested. Mammalian expression vectorsencoding dimer2, tdimer2(12) and mRFP1 were expressed in transientlytransfected HeLa cells. Within 12 hours the cells displayed strong redfluorescence evenly distributed throughout the nucleus and cytoplasm(data not shown).

In view of this result, it was tested whether an RFP-fusion polypeptidecould be created, where the RFP moiety retains its fluorescence, andwhere the fused polypeptide partner retains a native biologicalactivity. This experiment was conducted using the gap junction proteinconnexin43 (Cx43), which could demonstrate the advantage of a monomericred fluorescent protein if the fused Cx43 polypeptide retained itsbiological activity. A series of constructs consisting of Cx43 fused toeither GFP, T1, dimer2, tdimer2(12) or mRFP1 were expressed in HeLacells, which do not express endogenous connexins. Followingtransfection, the red fluorescence of the cells was observed with afluorescence microscope. The results of this experiment are shown inFIGS. 6A, 6C and 6E. As previously reported (Lauf et al., FEBS Lett.498:11-15 [2001]), the Cx43-GFP fusion protein was properly traffickedto the membrane and was assembled into functional gap junctions (datanot shown), whereas the Cx43-DsRed tetramer (i.e., the T1 tetramer)consistently formed perinuclear localized red fluorescent aggregates(FIG. 6E). Both Cx43-tdimer2(12) (not shown) and Cx43-dimer2 (FIG. 6C)were properly trafficked to the membrane though neither construct formedvisible gap junctions. In contrast, the Cx43-mRFP1 construct behavedidentically to Cx43-GFP and many red gap junctions were observed (FIG.6A).

In another experiment, the transfected cells were microinjected withlucifer yellow to assess the functionality of the gap junctions (seeFIGS. 6B, 6D and 6F; and FIG. 13). The Cx43-mRFP1 gap junctions rapidlyand reliably passed dye (FIG. 6B), while neither Cx43-T1 transfectedcells (FIG. 6E) nor non-transfected cells (not shown) passed dye. BothCx43-dimer2 and Cx43-tdimer2(12) constructs slowly passed dye to acontacting transfected neighbor about one third of the time (FIG. 6D).

The above-described results demonstrate that the monomeric mRFP1simultaneously overcomes the three critical problems associated with thewild-type tetrameric form of DsRed. Specifically mRFP1 is a monomer, itmatures rapidly, and it has minimal emission when excited at wavelengthsoptimal for GFP. These features make mRFP1 a suitable red fluorescentprotein for the construction of fusion proteins and multi-color labelingin combination with GFP. As demonstrated with the gap junction formingprotein Cx43, mRFP1 fusion proteins are functional and trafficked in amanner identical to their GFP analogues.

Although the extinction coefficient and fluorescence quantum yieldresult in reduced brightness of fully mature mRFP1 compared to DsRed,this is not an obstacle to use of mRFP1 in imaging experiments, as thereduced brightness is more than compensated for by the greater than10-fold decrease in maturation time for mRFP1. Variant RFP polypeptidesof the present invention, for example tdimer2(12), also find use as FRETbased sensors. This species is sufficiently bright, and displays FRETwith all variants of −4equorea GFP.

The present invention provides methods for the generation of stillfurther advantageous RFP species. These methods use multistepevolutionary strategies involving one or multiple rounds of evolutionwith few mutational steps per cycle. These methods also find use in theconverting of other oligomeric fluorescent proteins into advantageousmonomeric or dimeric forms.

Example 2 Preparation and Characterization of Fluorescent ProteinVariants

This example demonstrates that mutations can be introduced into GFPspectral variants that reduce or eliminate the ability of the proteinsto oligomerize.

ECFP (SEQ ID NO: 11) and EYFP-V68L/Q69K (SEQ ID NO: 12) at the dimerinterface were subcloned into the bacterial expression vector pRSETB(Invitrogen Corp., La Jolla Calif.), creating an N-terminal His6 tag onthe of ECFP (SEQ ID NO: 11) and EYFP-V68L/Q69K (SEQ ID NO: 12), whichallowed purification of the bacterially expressed proteins on anickel-agarose (Qiagen) affinity column. All dimer-related mutations inthe cDNAs were created by site-directed mutagenesis using theQuickChange mutagenesis kit (Stratagene), then expressed and purified inthe same manner. All cDNAs were sequenced to ensure that only thedesired mutations existed.

EYFP-V68L/Q69K (SEQ ID NO: 12) was mutagenized using the QuickChange kit(Stratagene). The overlapping mutagenic primers were designated “top”for the 5′ primer and “bottom” for the 3′ primer and are designatedaccording to the particular mutation introduced (see TABLE 1). Allprimers had a melting temperature greater than 70° C. The mutations weremade as close to the center of the primers as possible and all primerswere purified by polyacrylamide gel electrophoresis. The primers areshown in a 5′ to Y orientation, with mutagenized codons underlined(TABLE 1).

TABLE 1 SEQ ID Primer Sequence NO: A206K top CAG TCC AAG CTG AGC AAA GACCCC 25 AAC GAG AAG CGC GAT CAC A206K GTG ATC GCG CTT CTC GTT GGG GTC 26bottom TTT GCT CAG CTT GGA CTG L221K top CAC ATG GTC CTG AAG GAG TTC GTG27 ACC GCC GCC GGG L221K CCC GGC GGC GGT CAC GAA CTC CTT 28 bottom CAGGAC CAT GTG F223R top CAC ATG GTC CTG CTG GAG CGC GTG 29 ACC GCC GCC GGGF223R CCC GGC GGC GGT CAC GCG CTC CAG 30 bottom CAG GAC CAT GTGL221K/F223R CAC ATC GTC CTG AAG GAG CGC GTG 31 top ACC GCC GCC GGGL221K/F223R CCC GGC GGC GGT CAC GCG CTC CTT 32 bottom CAG GAC CAT GTG

For protein expression, plasmids containing cDNAs for the variousEYFP-V68L/Q69K (SEQ ID NO: 12) mutants were transformed into E. colistrain JM109 and grown to an OD₆₀₀ of 0.6 in LB containing 100 μg/mlampicillin at which time they were induced with 1 mM isopropylβ-D-thiogalactoside. The bacteria were allowed to express the protein atroom temperature for 6 to 12 hr, then overnight at 4° C., then werepelleted by centrifugation, resuspended in phosphate buffered saline (pH7.4), and lysed in a French press. Bacterial lysates were cleared bycentrifugation at 30,000×g for 30 min. The proteins in the clearedlysates were affinity-purified on Ni-NTA-agarose (Qiagen).

All GFPs used in these experiments were 238 amino acids in length.Subcloning the cDNAs encoding the GFPs into pRSETB resulted in thefusion of an additional 33 amino acids to the N-terminus of the GFPs.The sequence of this tag is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDP (SEQ IDNO: 22). Thus, the total length of the EYFP-V68L/Q69K (SEQ ID NO: 12)mutants expressed from this cDNA was 271 amino acids. The His₆ tag wasremoved using EKMax (Invitrogen) to determine if the associativeproperties measured for the GFPs were affected by the presence of theN-terminal His₆-tag. A dilution series of the enzyme and His₆-tagged GFPwas made to determine the conditions necessary for complete removal ofthe His₆-tag. The purity of all expressed and purified proteins wasanalyzed by SDS-PAGE. In all cases, the expressed proteins were verypure, with no significant detectable contaminating proteins, and allwere of the proper molecular weight. In addition, removal of the His₆tag was very efficient, as determined by the presence of a single bandmigrating at the lower molecular weight than the His₆-EYFP-V68L/Q69K.

Spectrophotometric analysis of the purified proteins determined thatthere was no significant change in either the extinction coefficient asmeasured by chromophore denaturation (Ward et al., In Green FluorescentProtein: Properties, Applications and Protocols,” eds. Chalfie and Kain,Wiley-Liss [1998]) or quantum yield (the standard used forEYFP-V68L/Q69K and the mutants derived therefrom was fluorescein) ofthese proteins with respect to EYFP-V68L/Q69K (SEQ ID NO: 12; “wtEYFP”;Table 2). Fluorescence spectra were taken with a Fluorologspectrofluorimeter. Absorbance spectra of proteins were taken with aCary UV-Vis spectrophotometer. Extinction coefficients were determinedby the denatured chromophore method (Ward et al., In Green FluorescentProtein: Properties, Applications and Protocols,” eds. Chalfie and Kain,Wiley-Liss [1998]).

TABLE 2 Quantum Extinction Protein Yield Coefficient WtEYFP 0.71*62,000* His₆ wtEYFP 0.67 67,410 His₆ wtEYFP L221K 0.67 64,286 His₆wtEYFP F223R 0.53 65,393 His₆ wtEYFP A206K 0.62 79,183 *published data(Cubitt et al., 1997)

To determine the degree of homoaffinity of the dimers, wtEYFP and thedimer mutants derived therefrom were subjected to sedimentationequilibrium analytical ultracentrifugation. Purified, recombinantproteins were dialyzed extensively against phosphate buffered saline (pH7.4), and 125 μl samples of protein at concentrations ranging from 50 μMto 700 μM were loaded into 6-channel centrifugation cells with EPONcenterpieces. Samples were blanked against the corresponding dialysisbuffer. Sedimentation equilibrium experiments were performed on aBeckman Optima XL-I analytical ultracentrifuge at 20° C. measuringradial absorbance at 514 nm. Each sample was examined at three or moreof the following speeds: 8,000 rpm, 10,000 rpm, 14,000 rpm, and 20,000rpm. Periodic absorbance measurements at each speed ensured that thesamples had reached equilibrium at each speed.

The data were analyzed globally at all rotor speeds by nonlinearleast-squares analysis using the software package (Origin) supplied byBeckman. The goodness of fit was evaluated on the basis of the magnitudeand randomness of the residuals, expressed as the difference between theexperimental data and the theoretical curve and also by checking each ofthe tit parameters for physical reasonability. The molecular weight andpartial specific volume of each protein were determined using Sedenterpv 1.01, and the data were factored into the equation for thedetermination of homoaffinity (TABLE 3).

TABLE 3 Molecular Partial Mutant Weight Specific Volume WtEYFP 26796.230.7332 His₆ wtEYFP 30534.26 0.7273 His₆ EYFP A206K 30593.37 0.7277 EYFPL221K 30551.29 0.7270 His₆ EYFP L221K 30549.27 0.7271 His₆ EYFP F223R30543.27 0.7270 His₆EYFP L221K/F223R 30560.30 0.7267

In addition, dissociation constants (K_(d)) derived from the datagenerated by analytical ultracentrifugation are shown for some proteins(TABLE 4).

TABLE 4 Protein K_(d)(mM) His6 wtEYFP 0.11 His6 wtEYFP L221K 9.7 His6wtEYFP F223R 4.8 His6 wtEYFP A206K 74 His6 wtEYFP L221K/F223R 2.4

For experiments in living cells, ECFP (SEQ ID NO: 11; “wtECFP”) andEYFP-V68L/Q69K (SEQ ID NO: 12; “wtEYFP”) targeted to the plasma membrane(PM) were subcloned into the mammalian expression vector, pcDNA3(Invitrogen Corp.) and mutagenized and sequenced as described above.Targeting of the GFP variants to the PM was accomplished by makingeither N-terminal or C-terminal fusions of the GFP variant to shortpeptides containing a consensus sequence for acylation and/orprenylation (post-translational lipid modifications). The cDNAs of thePM targeted GFP variants were transfected and expressed in either HeLacells or MDCK cells, and the expression pattern and degree ofassociation were determined using fluorescent microscopy. FRETefficiency was measured to determine the degree of interaction of thePM-ECFP and PM-EYFP-V68L/Q69K. Analysis of the interactions by the FRETdonor-dequench method (Miyawaki and Tsien, supra, 2000) demonstratedthat the wtECFP and wtEYFP interacted in a manner that was dependentupon the association of the wtECFP and wtEYFP, and that this interactionwas effectively eliminated by changing the amino acids in thehydrophobic interface to any one or a combination of the mutationsA206K, L221K and F223R.

These results demonstrate that the solution oligomeric state of AequoreaGFP and its spectral variants, and dimer mutants derived therefrom, wereaccurately determined by analytical ultracentrifugation. The ECFP (SEQID NO: 11) and EYFP-V68L/Q69K (SEQ ID NO: 12) GFP spectral variantsformed homodimers with a fairly high affinity of about 113 μM. By usingsite directed mutagenesis, the amino acid composition was altered so asto effectively eliminate dimerization and the cell biological problemsassociated with it. Thus, the modified fluorescent proteins provide ameans to use FRET to measure the associative properties of host proteinsfused to the modified CFP or YFP. The ambiguity and potential for falsepositive FRET results associated with ECFP (SEQ ID NO: 11) andEYFP-V68L/Q69K (SEQ ID NO: 12) dimerization have been effectivelyeliminated, as has the possibility of misidentification of thesubcellular distribution or localization of a host protein due todimerization of GFPs.

The-Renilla GFP and the Discosomd red fluorescent protein are obligateoligomers in solution. Because it was generally believed that AequoreaGFP could also dimerize in solution, and because GFP crystallizes as adimer, the present investigation was designed to characterize theoligomeric state of GFP. The crystallographic interface between the twomonomers included many hydrophilic contacts as well as severalhydrophobic contacts (Yang et al., supra, 1996). It was not immediatelyclear, however, to what degree each type of interaction contributed tothe formation of the dimer in solution.

As disclosed herein, the extent of GFP self-association was examinedusing sedimentation equilibrium, analytical ultracentrifugation, whichis very useful for determining the oligomeric behavior of molecules bothsimilar (self associating homomeric complexes) and dissimilar(heteromeric complexes; see Laue and Stafford, Ann. Rev. Biophys.Biomol. Struct. 28:75-100, 1999). In contrast to X-ray crystallography,the experimental conditions used in the analytical ultracentrifugationexperiments closely approximated cellular physiological conditions.Monomer contact sites identified by X-ray crystallography within amultimeric complex are not necessarily the same as those in solution.Also in contrast to analytical ultracentrifugation, X-raycrystallography alone cannot provide definitive information about theaffinity of the complex. The results of this investigation demonstratethat replacement of the hydrophobic residues A206, L221 and F223 withresidues containing positively charged side chains (A206K, L221K andF223R) eliminated dimerization as determined by analyticalultracentrifugation in vitro and by analysis of the concentrationdependence of FRET in intact cells.

Example 3 Characterization of the Coral Red Fluorescent Protein, DsRed,and Mutants Thereof

This example describes the initial biochemical and biologicalcharacterization of DsRed and DsRed mutants.

The coding sequence for DsRed was amplified from pDsRed-N1 (ClontechLaboratories) with PCR primers that added an N terminal BamHIrecognition site upstream of the initiator Met codon and a C terminalEco RI site downstream of the STOP codon. After restriction digestion,the PCR product was cloned between the Bam HI and Eco RI sites ofpRSET_(B) (Invitrogen), and the resulting vector was amplified in DH5αbacteria. The resulting plasmid was used as a template for error-pronePCR (Heim and Tsien, Curr. Biol. 6:178-182, 1996, which is incorporatedherein by reference) using primers that were immediately upstream anddownstream of the DsRed coding sequence, theoretically allowing mutationof every coding base, including the initiator Met. The ″ mutagenized PCRfragment was digested with Eco RI and Bam HI and recloned intopRSET_(B). Alternatively, the Quick-Change mutagenesis kit (Stratagene)was used to make directed mutations on the pRSET_(B)-DsRed plasmid.

In both random and directed mutagenesis studies, the mutagenized plasmidlibrary was electroporated into JM109 bacteria, plated on LB platescontaining ampicillin, and screened on a digital imaging device (Bairdet al., Proc. Natl. Acad. Sci., USA 96:11242-11246, 1999, which isincorporated herein by reference). This device illuminated plates withlight from a 150 Watt xenon arc lamp, filtered through bandpassexcitation filters and directed onto the plates with two fiber opticbundles. Fluorescence emission from the plates was imaged throughinterference filters with a cooled CCD camera. Images taken at differentwavelengths could be digitally ratioed using MetaMorph software(Universal Imaging) to allow identification of spectrally shiftedmutants. Once selected, the mutant colonies were picked by hand intoLB/Amp medium, after which the culture was used for protein preparationor for plasmid preparations. The DsRed mutant sequences were analyzedwith dye-terminator dideoxy sequencing.

DsRed and its mutants were purified using the N-terminal polyhistidinetag (SEQ ID NO: 22; see Example 1) provided by the pRSETB expressionvector (see Baird et al., supra, 1999). The proteins weremicroconcentrated and buffer exchanged into 10 mM Tris (pH 8.5) using aMicrocon-30 (Amicon) for spectroscopic characterization. Alternatively,the protein was dialyzed against 10 mM Tris (pH 7.5) for oligomerizationstudies because microconcentration resulted in the production of largeprotein aggregates. To test for light sensitivity of protein maturation,the entire synthesis was repeated in the dark, with culture flaskswrapped in foil, and all purification was performed in a room that wasdimly lit with red lights. There was no difference in protein yield orcolor when the protein was prepared in light or dark.

Numbering of amino acids conforms to the wild type sequence of drFP583(DsRed; Matz et al., Nature Biotechnology 17:969-973 [1999]), in whichresidues 66-68, Gln-Tyr-Gly, are homologous to the chromophore-formingresidues (65-67, Ser-Tyr-Gly) of GFP. The extra amino acid introduced byClontech after the initiator Met was numbered “1a” and the residues ofthe N-terminal polyhistidine tag were numbered −33 to −1.

Fluorescence spectra were taken with a Fluorolog spectrofluorimeter.Absorbance spectra of proteins were taken with a Cary UV-Visspectrophotometer. For quantum yield determination, the fluorescence ofa solution of DsRed or DsRed K83M in phosphate buffered saline wascompared to equally absorbing solutions of Rhodamine B and Rhodamine 101in ethanol. Corrections were included in the quantum yield calculationfor the refractive index difference between ethanol and water. Forextinction coefficient determination; native protein absorbance wasmeasured with the spectrophotometer, and protein concentration wasmeasured by the BCA method (Pierce).

The pH sensitivity of DsRed was determined in a 96 well format by adding100 μL of dilute DsRed in a weakly buffered solution to 100 μL ofstrongly buffered pH solutions in triplicate (total 200 μL per well) forpH 3 to pH 12. The fluorescence of each well was measured using a525-555 nm bandpass excitation filter and a 575 nm long pass emissionfilter. After the 96 well fluorimeter measurements were taken, 100 μl ofeach pH buffered DsRed solution was analyzed on the spectrofluorimeterto observe pH-dependent spectral shape changes. For time-trials of DsRedmaturation, a dilute solution of freshly synthesized and purified DsRedwas made in 10 mM Tris (pH 8.5), and this solution was stored at roomtemperature in a stoppered cuvette (not airtight) and subjected toperiodic spectral analysis. For mutant maturation data, fluorescenceemission spectra (excitation at 475 nm or 558 nm) were taken directlyafter synthesis and purification, and then after more than 2 monthsstorage at 4° C. or at room temperature.

Quantum yields for photodestruction were measured separately on amicroscope stage or in a spectrofluorimeter. Microdroplets of aqueousDsRed solution were created under oil on a microscope slide and bleachedwith 1.2 W/cm² of light through a 525-555 nm bandpass filter.Fluorescence over time was monitored using the same filter and a 563-617nm emission filter. For comparison, EGFP (containing mutations F64L,S65T; SEQ ID NO: 13) and EYFP-V68L/Q69K (also containing mutations S65G,S72A, T203Y; SEQ ID NO: 12) microdroplets were similarly bleached with1.9 W/cm² at 460-490 nm while monitoring at 515-555 and 523-548 nm,respectively.

For the spectrofluorimeter bleaching experiment, a solution of DsRed wasprepared in a rectangular microcuvette and overlaid with oil so that theentire 50 μL of protein solution resided in the 0.25 cm×0.2 cm×1 cmillumination volume. The protein solution was illuminated with 0.02W/cm² light from the monochromator centered at 558 nm (5 nm bandwidth).Fluorescence over time was measured at 558 nm excitation (1.25 nmbandwidth) and 583 nm emission. Quantum yields (Φ) for photobleachingwere deduced from the equation Φ=(ε·I·t_(90%))⁻¹, where ε is theextinction coefficient in cm²mol⁻¹, I is the intensity of incident lightin einsteins cm⁻²s⁻¹ and t_(90%) is the time in seconds for thefluorophore to be 90% bleached (Adams et al., J. Am. Chem. Soc.110:3312-3320, 1988, which is incorporated herein by reference).

Polyhistidine-tagged DsRed, DsRed K83M and wild type Aequorea GFP (SEQID NO: 10) were nm on a 15% polyacrylamide gel without denaturation. Toprevent denaturation, protein solutions (in 10 mM Tris HCl, pH 7.5) weremixed 1:1 with 2×SDS-PAGE sample buffer (containing 200 mMdithiothreitol) and loaded directly onto the gel ″ without boiling. Abroad range pre-stained molecular weight marker set (BioRad) was used asa size standard. The gel was then imaged on an Epson 1200 Perfectionflatbed scanner.

Purified recombinant DsRed was dialyzed extensively against phosphatebuffered saline (pH 7.4) or 10 mM Tris, 1 mM EDTA (pH 7.5).Sedimentation equilibrium experiments were performed on a Beckman OptimaXL-I analytical ultracentrifuge at 20° C. measuring absorbance at 558 nmas a function of radius. 125 μL samples of DsRed at 3.57 μM (0.25absorbance units) were loaded into 6 channel cells. The data wereanalyzed globally at 10,000, 14,000, and 20,000 rpm by nonlinearleast-squares analysis using the Origin software package (Beckman). Thegoodness of fit was evaluated on the basis of the magnitude andrandomness of the residuals, expressed as the difference between theexperimental data and the theoretical curve and also by checking each ofthe fit parameters for physical reasonability.

FRET between immature green and mature red DsRed was examined inmammalian cells. DsRed in the vector pcDNA3 was transfected into HeLacells using Lipofectin, and 24 hr later the cells were imaged on afluorescence microscope. The fluorescences of the immature green.species (excitation 465-495 nm, 505 nm dichroic, emission 523-548 nm)and of mature red protein (excitation 529-552 nm, 570 nm dichroic,emission 563-618 nm) were measured with a cooled CCD camera. Thesemeasurements were repeated after selective photobleaching of the redcomponent by illumination with light from the xenon lamp, filtered onlyby the 570 nm dichroic, for cumulative durations of 3, 6, 12, 24, and 49min. By the final time, about 95% of the initial red emission haddisappeared, whereas the green emission was substantially enhanced.

Yeast two hybrid assays were also performed. The DsRed coding region wascloned in-frame downstream of the Gal4 activation domains (the “bait”;amino acid residues 768-881) and DNA binding domains (the “prey”; aminoacid residues 1-147) in the pGAD GH and pGBT9 vectors, respectively,(Clontech). These DsRed two hybrid plasmids were transformed into theHF7C strain of S. cerevisiae, which cannot synthesize histidine in theabsence of interaction between the proteins fused to the Gal4 fragments.Yeast containing both DsRed-bait and DsRed-prey plasmids were streakedon medium lacking histidine and assayed for growth by visuallyinspecting the plates. Alternatively, the yeast were grown on filtersplaced on plates lacking tryptophan and leucine to select for the baitand prey plasmids. After overnight growth, the filters were removed fromthe plates, frozen in liquid nitrogen, thawed, and incubated in X-galovernight at 30° C. and two days at 4° C. to test for β-galactosidaseactivity (assayed by blue color development). In both theβ-galactosidase and histidine growth assays, negative controls consistedof yeast containing bait and prey plasmids, but only the bait or theprey was fused to DsRed.

Surprisingly, DsRed took days at room temperature to reach full redfluorescence. At room temperature, a sample of purified proteininitially showed a major component of green fluorescence (excitation andemission maxima at 475 and 499 nm, respectively), which peaked inintensity at about 7 hr and decreased to nearly zero over two days.Meanwhile, the red fluorescence reached half its maximal fluorescenceafter approximately 27 hr and required more than 48 hr to reach greaterthan 90% of maximal fluorescence (see Baird et al., supra, 2000).

Fully matured DsRed had an extinction coefficient of 75,000 M⁻¹cm⁻¹ atits 558 nm absorbance maximum and a fluorescence quantum yield of 0.7,which is much higher than the values of 22,500 M⁻¹cm⁻¹ and 0.23previously reported (Matz et al., Nature Biotechnology 17:969-973[1999]). These properties make mature DsRed quite similar to rhodaminedyes in wavelength-and brightness. Unlike most GFP variants, DsReddisplayed negligible (<10%) pH-dependence of absorbance or fluorescencefrom pH 5 to 12. (see Baird et al., supra, 2000). However, acidificationto pH 4-4.5 depressed both the absorbance and excitation at 558 nmrelative to the shorter wavelength shoulder at 526 nm, whereas theemission spectrum was unchanged in shape. DsRed was also relativelyresistant to photobleaching. When exposed to a beam of 1.2 W/cm² ofapproximately 540 nm light in a microscope stage, microdroplets of DsRedunder oil took 1 hr to bleach 90%, whereas 20 mW/cm² of 558 nm light ina spectrofluorimeter microcuvette required 83 hr to bleach 90%. Themicroscope and fluorimeter measurements, respectively, gave photobleachquantum efficiencies of 1.06×10⁻⁶ and 4.8×10⁻⁷ (mean of 7.7×10⁻⁷).Analogous microscope measurements of EGFP (S65T; SEQ ID NO: 13) andEYFP-V68L/Q69K (SEQ ID NO: 12; including Q69K) gave 3×10⁻⁶ and 5×10⁻⁵,respectively.

In an effort to examine the nature of the red chromophore and toidentify DsRed variants useful as biological indicators, DsRed wasmutagenized randomly and at specific sites predicted by sequencealignment with GFP to be near the chromophore. Many mutants that maturedmore slowly or not at all were identified, but none were identified thatmatured faster than DsRed. Screening of random mutants identifiedmutants that appeared green or yellow, which was found to be due tosubstitutions K83E, K83R, S197T, and Y120H. The green fluorescence wasdue to a mutant species with excitation and emission maxima at 475 and500 nm, respectively, whereas the yellow was due to a mixture of thisgreen species with DsRed-like material, rather than to a single speciesat intermediate wavelengths.

The DsRed K83R mutant had the lowest percentage conversion to red inthis series of experiments, and proved very useful as a stable versionof the immature green-fluorescing form of DsRed (see Baird et al.,supra, 2000). Further directed mutagenesis of K83 yielded more green andyellow mutants that were impaired in chromophore maturation. In many ofthe K83 mutants that matured slowly and incompletely, the red peak wasat longer wavelengths than DsRed. K83M was particularly interestingbecause its final red-fluorescing species showed a 602 nm emissionmaximum, with relatively little residual green fluorescence and arespectable quantum yield, 0.44. However, its maturation was slower thanthat of the wild type DsRed. Y120H had a red shift similar to that ofK83M and appeared to produce brighter bacterial colonies, but alsomaintained much more residual green fluorescence.

Spectroscopic data of the DsRed mutants are shown in FIG: 15. In thisFIG., “maturation” of protein refers to the rate of appearance of thered fluorescence over the two days after protein synthesis. Because somematuration occurs during the synthesis and purification (which take 1-2days), numerical quantification is not accurate. A simple +/−ratingsystem was used, wherein (−−) means very little change, (−) means a 2-5fold increase in red fluorescence, (+) means 5-20 fold increase, and(++) indicates the wild type increase (approximately 40 fold). The redgreen ratio was determined two months after protein synthesis bydividing the peak emission fluorescence obtained at 558 nm excitation bythe 499 nm fluorescence obtained at 475 nm excitation from the samesample. This does not represent a molar ratio of the two species becausethe ratio does not correct for differences in extinction coefficient orquantum yields between the two species, or the possibility of FRETbetween the two species if they are in a macromolecular complex.

To determine whether Lys70 or Arg95 can form imines with the terminalcarbonyl of a GFP-like chromophore (see Tsien, Nature Biotechnol.,17:956-957, 1999), DsRed mutants K70M, K70R, and R95K were produced.K70M remained entirely green with no red component, whereas K70R maturedslowly to a slightly red-shifted red species. The spectral similarity ofK70R to wild type DsRed argues against covalent incorporation of eitheramino acid into the chromophore. No fluorescence at any visiblewavelength was detected from R95K, which might be expected because Arg95is homologous to Arg96-of GFP, which is conserved in all fluorescentproteins characterized to date (Matz et al., Nature Biotechnology17:969-973 [1999]). The failure of R95K to form a green chromophoreprevented testing whether Arg95 was also required for reddening.

In view of the propensity of Aequorea GFP to form dimers at highconcentrations in solution and in some crystal forms, and the likelihoodthat Renilla GFP forms an obligate dimer (Ward et al., In GreenFluorescent Protein: Properties, Applications and Protocols,” eds.Chalfie and Kain, Wiley-Liss [1998]), the ability of DsRed tooligomerize was examined. Initial examination of the expressed proteinsby SDS-PAGE suggested that aggregates formed, in thatpolyhistidine-tagged proteins DsRed and DsRed K83R migrated as red andyellow-green bands, respectively, at an apparent molecular weight ofgreater than 110 kDa when mixed with 200 mM DTT and not heated beforeloading onto the gel (see Baird et al., supra, 2000). In comparison,Aequorea GFP, when treated similarly, ran as a fluorescent green bandnear its predicted monomer molecular weight of 30 kDa. The highmolecular weight DsRed band was not observed when the sample was brieflyboiled before electrophoresis (see Gross et al., supra, 2000). Underthese conditions, a band near the predicted monomer molecular weight of30 kDa predominated and was colorless without Coomassie staining.

To determine the oligomerization status more rigorously, the DsRedprotein was subjected to analytical equilibrium centrifugation (Laue andStafford, supra, 1999). Global curve fitting of the absorbance datadetermined from the radial scans of equilibrated DsRed indicated thatDsRed exists as an obligate tetramer in solution (Baird et al., supra,2000), in both low salt and physiological salt concentrations. When thedata was modeled with a single-species tetramer, the fitted molecularweight was 119,083 Da, which is in excellent agreement with thetheoretical molecular weight of 119,068 Da for the tetramer ofpolyHis-tagged DsRed. Attempts to fit the curves with alternativestoichiometries from monomer to pentamer failed to converge or gaveunreasonable values for the floating variables and large, non-randomresiduals. The residuals for the tetramer fit were much smaller and morerandomly distributed, but were somewhat further improved by extendingthe model to allow the obligate tetramer to dimerize into an octamer,with a fitted dissociation constant of 39 μM. Thus the 558-nm-absorbingspecies appears to be tetrameric over the range of monomerconcentrations from 14 nM to 11 μM in vitro. The hint of octamerformation at the highest concentrations is only suggestive because thehighest concentrations of tetramer achieved in the ultracentrifugationcell remained more than an order of magnitude below the fitteddissociation constant.

To confirm whether DsRed also oligomerizes in live cells, FRET analysiswas performed in mammalian cells and in two hybrid assays in yeastcells. HeLa cells were transfected with wild type DsRed and imaged 24 hrlater, when they contained a mixture of the immature green intermediateand the final red form. The green fluorescence was monitoredintermittently before and during selective photobleaching of the redspecies over 49 min of intense orange illumination. If the two proteinswere non-associated, bleaching the red species would be expected to haveno effect on the green fluorescence. In fact, however, the greenfluorescence increased by 2.7 to 5.8 fold in different cells,corresponding to FRET efficiencies of 63% to 83%. These values equal orsurpass the highest FRET efficiencies ever observed between GFP mutants,68% for cyan and yellow fluorescent proteins linked by a zincion-saturated zinc finger domain (Miyawaki and Tsien, supra, 2000).

Additional evidence of in vivo oligomerization was provided by thedirected yeast two hybrid screen. When DsRed fusions to the Gal4 DNAbinding domain and activation domain were expressed in HF7C yeast, theyeast demonstrated a his' phenotype and were able to grow withoutsupplemental histidine, indicating a two hybrid interaction hadoccurred. Neither fusion construct alone (DsRed-DNA binding domain orDsRed-activation domain) produced the his⁺ phenotype, indicating that aDsRed-DsRed interaction, and not a non-specific DsRed-Gal4 interaction,was responsible for the positive result. In addition, the his⁺ yeastturned blue when lysed and incubated with X-gal, suggesting that theDsRed-DsRed interaction also drove transcription of the θ-galactosidasegene. Thus, two separate transcriptional measurements of the yeast twohybrid assay confirmed that DsRed associates in vivo.

This investigation of DsRed revealed that DsRed has many desirableproperties, as well as some nonoptimal properties, with respect to itsbeing useful to complement or as an alternative to GFPs. One of the mostimportant favorable properties identified was that DsRed has a muchhigher extinction coefficient and fluorescence quantum yield (0.7) thanwas previously reported, such that the fluorescence brightness of themature well-folded protein is comparable to rhodamine dyes and to thebest GFPs.

DsRed also is quite resistant to photobleaching by intensities typicalof spectrofluorimeters (mW/cm²) or microscopes with arc lampillumination and interference filters (W/cm²), showing a photobleachingquantum yield on the order of 7×10⁻⁷ in both regimes. This value issignificantly better than those for two of the most popular green andyellow GFP mutants, EGFP (3×10⁻⁶) and EYFP-V68L/Q69K (5×10⁻⁵). The meannumber of photons that a single molecule can emit before photobleachingis the ratio of the fluorescence and photobleaching quantum yields, or1×10⁶, 2×10⁵, and 1.5×10⁴ for DsRed, EGFP, and EYFP-V68L/Q69K,respectively. A caveat is that the apparent photobleaching quantum yieldmight well increase at higher light intensities and shorter times if themolecule can be driven into dark states such as triplets or tautomersfrom which it can recover its fluorescence. GFPs usually show a range ofsuch dark states (Dickson et al., Nature 388:355-358, 1997; Schwille etal., Proc. Natl. Acad. Sci., USA 97:151-156, 2000), and there is noreason to expect that DsRed will be any simpler. The photobleachingmeasurements described herein were made over minutes to hours, andinclude ample time for such recovery. In contrast, fluorescencecorrelation spectroscopy and flow cytometry monitor single passages ofmolecules through a focused laser beam within microseconds tomilliseconds, such that temporary dark states that last longer than thetransit time count as photobleaching, raising the apparent quantum yieldfor bleaching. Techniques such as laser scanning confocal microscopy, inwhich identified molecules are repetitively scanned, will showintermediate degrees of photobleaching depending on the time scale ofillumination and recovery.

Another desirable feature of DsRed is its negligible sensitivity to pHchanges over the wide range (pH 4.5 to 12). The currently availablebrighter GFP mutants are more readily quenched than DsRed by acidic pH.Such pH sensitivity can be exploited under controlled conditions tosense pH changes, especially inside organelles or other specificcompartments (see Llopis et al., Proc. Natl. Acad. Sci., USA95:6803-6808, 1998), although this feature can cause artifacts in someapplications.

DsRed mutants such as K83M demonstrate that DsRed can be pushed tolonger wavelengths (564 and 602 nm excitation and emission maxima),while retaining adequate quantum efficiency (0.44). The 6 nm and 19 nmbathochromic shifts correspond to 191 cm⁻¹ and 541 cm⁻¹ in energy, whichare of respectable magnitude for a single amino acid change that doesnot modify the chromophore. A homolog of DsRed recently cloned from asea anemone has an absorbance maximum at 572 nm and extremely weakemission at 595 nm with quantum yield <0.001; one mutant had an emissionpeak at 610 nm but was very dim and slow to mature (Lukyanov et al., J.Biol. Chem. 275:25879-25882, 2000, which is incorporated herein byreference).

Less desirable features of DsRed include its slow and incompletematuration, and its capacity to oligomerize. A maturation time on theorder of days precludes a use of DsRed as a reporter for short term geneexpression studies and for applications directed to tracking fusionproteins in organisms that have short generation times or fastdevelopment. Since maturation of GFPs was considerably accelerated bymutagenesis (Heim et al., Nature 373:663-664, 1995, which isincorporated herein by reference), DsRed similarly can be mutagenizedand variants having faster maturation times can be isolated.

Because the Lys83 mutants all permitted at least some maturation, it isunlikely that the primary amine plays a direct catalytic role for thisresidue, a suggestion supported by the observation that the mostchemically conservative replacement, Lys to Arg, impeded red developmentto the greatest extent. Ser197 provided a similar result, in that themost conservative possible substitution, Ser to Thr, also significantlyslowed maturation. Mutations at the Lys83 and Ser197 sites appearedseveral times independently in separate random mutagenesis experimentsand, interestingly, Lys83 and Ser197 are replaced by Leu and Thr,respectively, in the highly homologous cyan fluorescent protein dsFP483from the same Discosoma species. Either of the latter two mutationscould explain why dsFP483 never turns red. Residues other than Lys83 andSer197 also affected maturation to the red.

The multimeric nature of DsRed was demonstrated by four separate linesof evidence, including slow migration on SDS-PAGE unless pre-boiled,analytical ultracentrifugation, strong FRET from the immature green tothe final red form in mammalian cells, and directed two hybrid assays inyeast using HIS3 and LacZ reporter genes. Analytical ultracentrifugationprovided the clearest evidence for an obligate stoichiometry of fourover the entire range of monomer concentrations assayed (10⁻⁸ to 10⁻⁵M), with a hint that octamer formation can occur at yet higherconcentrations. In addition, the tests in live cells confirmed thataggregation occurs under typical conditions of use, including thereducing environment of the cytosol and the presence of native proteins.

While oligomerization of DsRed does not preclude its use as a reporterof gene expression, it can result in artifactual results in applicationswhere DsRed is fused to a host protein, for example, to report on thetrafficking or interactions of the host protein in a cell. For a hostprotein of mass M without its own aggregation tendencies, fusion withDsRed can result in the formation of a complex of at least 4(M+26 kDa).Furthermore, since many proteins in signal transduction are activated byoligomerization, fusion to DsRed and consequent association can resultin constitutive signaling. For host proteins that are oligomeric, fusionto DsRed can cause clashes of stoichiometry, steric conflicts ofquaternary structures, or crosslinking into massive aggregates. In fact,red chameleons, i.e., fusions of cyan fluorescent protein, calmodulin,and calmodulin-binding peptide, and DsRed, are far more prone to formvisible punctae in mammalian cells than the corresponding yellowchameleons with yellow fluorescent protein in place of DsRed (Miyawakiet al., Proc. Natl. Acad. Sci., USA 96:2135-2140, 1999).

The results disclosed above indicate that variants of DsRed, like thoseof the GFPs, can be produced such that the propensity of the fluorescentprotein to oligomerize is reduced or eliminated. DsRed variants can beconstructed and examined, for example, using a yeast two hybrid or othersimilar assay to identify and isolate non-aggregating mutants. Inaddition, the X-ray crystallographic structure of DsRed can be examinedto confirm that optimal amino acid residues are modified to produce aform of DsRed having a reduced propensity to oligomerize.

Example 4 DsRed Variants Having Reduced Propensity to Oligomerize

This example demonstrates that mutations corresponding to thoseintroduced into GFP variants to reduce or eliminate oligomerization alsocan be made in DsRed to reduce the propensity of DsRed to formtetramers.

In view of the results described in Example 1 and guided by the DsRedcrystal structure, amino acid residues were identified as potentiallybeing involved in DsRed oligomerization. One of these amino acids,isoleucine-125 (I125), was selected because, in the oligomer, the I125residues of the subunits were close to, each other in a pairwisefashion; i.e., the side chain of I125 of the A subunit was about 4Angstroms from the side chain of I125 of the C subunit, and the I125residues in the B and D subunits were similarly positioned. In addition,the area in which the I125 side chains reside exhibited hydrophobicity,analogous to that identified in Aequorea GFP variants, which wasdemonstrated to be involved in the inter-subunit interaction. Based onthese observations, DsRed mutants containing substitutions of positivelycharged amino acids, Lys (K) and Arg (R), for I125 were generated.

DsRed 11 25K and I125R were prepared with the QuickChange MutagenesisKit using the DsRed cDNA (SEQ ID NO: 23; Clontech) subcloned into theexpression vector pRSETB (Invitrogen) as the template for mutagenesis.The primers for mutagenesis, with the mutated codons underlined, were asfollows:

TABLE 5 Primer Sequence SEQ ID NO I125K 5′-TAC AAG GTG AAG TTC AAG GGC33 (forward) GTG AAC TTC CCC-3′ I125K 5′-GGG GAA GTT CAC GCC CTT GAA 34(reverse) CTT CAC CTT GTA-3′ I125R 5′-TAC AAG GTG AAG TTC CGC GGC 35(forward) GTG AAC TTC CCC-3′ I125R 5′-GGG GAA GTT CAC GCC GCG GAA 36(reverse) CTT CAC CTT GTA-3′

The mutant proteins were prepared following standard methodology andanalyzed with polyacrylamide gel electrophoresis as described (Baird etal., supra, 2000). For further analysis, DsRed I125R was dialyzedextensively in PBS, then diluted in PBS until the absorbance of thesolution at 558 nm was 0.1. This solution was centrifuged in a BeckmanXL-1 analytical ultracentrifuge in PBS at 10,000 rpm, 12,000 rpm, 14,000rpm, and 20,000 rpm. Absorbance at 558 nm versus radius was determinedand compared to a wild type tetrameric DsRed control (Baird et al.,supra, 2000).

The DsRed I125K yielded a protein that became red fluorescent and was amixture of dimer and tetramer as analyzed by non-denaturingpolyacrylamide gel electrophoresis of the native protein. The sameanalysis of DsRed I125R revealed that the protein was entirely dimeric.The dimeric status of DsRed I125R was confirmed by analyticalultracentrifugation; no residual tetramer was detected. These resultsdemonstrate that the interaction between the A:C subunits and the B:Dsubunits can be disrupted, thereby reducing the propensity of the DsRedvariant to oligomerize. No attempt was made to disrupt the A:B and C:Dinterfaces. These results demonstrate that the method of reducing oreliminating oligomerization of the GFP variants as described in Example1 is generally applicable to other fluorescent proteins that have apropensity to oligomerize.

Example 5 Preparation and Characterization of Tandem DsRed Dimers

This example demonstrates that a tandem DsRed protein can be formed bylinking two DsRed monomers, and that such tandem DsRed proteins maintainemission and excitation spectra characteristic of DsRed, but do notoligomerize.

To construct tandem DsRed (tDsRed), a 3′ primer,5′-CCGGATCCCCTTTGGTGCTGCCCTCTCCGCTGCCAGGCTTGCCGCTGCCGCTGGTGCTGCCAAGGAACAGATGGTGGCGTCCCTCG-3′ (SEQ ID NO: 37), was designed thatoverlapped the last 25 by of DsRed (derived from the Clontech vectorpDsRed-N1) and encoded for the linker sequence GSTSGSGKPGSGEGSTKG (SEQID NO: 38), followed by a Bam HI restriction site in frame with theBamHI site of pRSET_(B) (Invitrogen). It was later determined that theabove primer sequence contains three mismatches in the overlap regionand contained several codons that were not optimal for mammalianexpression. Accordingly, a new 3′ primer,5′-CCGGATCCCCCTTGGTGCTGCCCTCCCCGCTGCCGGGCTTCCCGCTCCCGCTGGTGCTGCCCAGGAACAGGTGGTGGCGGCCCTCG-3′ (SEQ ID NO: 39), also was used. The5′ primer, 5′-GTACGACGATGACGATAAGGATCC-3′ (SEQ ID NO: 40) also containeda Bam HI restriction site in frame with the Bam HI site of pRSET_(B).

PCR amplification of DsRed and of DsRed-I125R with the new linker wasaccomplished with Taq DNA polymerase (Roche) and an annealing protocolthat included 2 cycles at 40° C., 5 cycles at 43° C., 5 cycles at 45°C., and 15 cycles at 52° C. The resulting PCR product was purified byagarose gel-electrophoresis and digested with Bam HI (New EnglandBiolabs). Bam HI and calf intestinal phosphatase (New England Biolabs)treated vector was prepared from pRSETB with DsRed or DsRed-I125Rinserted in frame with the His-6 tag and between the 5′ Bam HI and 3′Eco RI restriction sites.

Following ligation of the digested PCR products and vector with T4 DNAligase (NEB), the mixture was used to transform competent E. coli DHSαby heat shock. Transformed colonies were grown on LB agar platessupplemented with the antibiotic ampicillin. Colonies were picked atrandom, and plasmid DNA was isolated through standard miniprepprocedures (Qiagen). DNA sequencing was used to confirm the correctorientation of the inserted sequence.

In order to express protein; the isolated and sequenced vectors wereused to transform competent E. coli JMI09(DE3). Single colonies grown onLB agar/ampicillin were used to inoculate 1 liter cultures ofLB/ampicillin, then were grown with shaking at 225 rpm and 37° C. untilthe broth reached an OD₆₀₀ of 0.5-1.0. IPTG was added to a finalconcentration of 100 mg/l and the culture was grown for either 5 hr at37° C. (tDsRed) or 24 hr at room temperature (RT; tDsRed-I125R). Cellswere harvested by centrifugation (10 min, 5000 rpm), the pellet wasresuspended in 50 mM Tris pH 7.5, and the cells were lysed by a singlepass through a French press. Protein was purified by Ni-NTA (Qiagen)chromatography as described by the manufacturer and was stored in theelution buffer or was dialyzed into 50 mM Tris, pH 7.5.

With respect to the excitation and emission spectra as well as thematuration time of tDsRed and tDsRed-I125R, the proteins behavedidentically to their untethered counterparts. As expected, tDsReddeveloped visible fluorescence within approximately 12 hr at roomtemperature, while tDsRed-I125R required several days before significantred color developed. The maturation of tDsRed-I125R continued for up toapproximately 10 days. The excitation and emission maxima were unchangedat 558 nm and 583 nm, respectively.

The differences in the tandem dimer became apparent when the proteinswere analyzed by SDS-polyacrylamide electrophoresis. Due to the highstability of the tetramer, DsRed that was not subjected to boilingmigrated with an apparent molecular mass of about 110 kDa. In addition,the band on the gel, which corresponded to a DsRed tetramer, retainedits red fluorescence, indicating that the rigid barrel structure of eachmonomer was intact. When the sample was boiled before loading, DsRed wasnon-fluorescent and presumably denatured, and ran as a monomer ofapproximately 32 kDa.

SDS-PAGE analysis confirmed the tandem structure of the expressed redfluorescent proteins, tDsRed and tDsRed-I125R. The unboiled tDsRedmigrated at the same apparent molecular mass (about 110 kDa) as unboilednormal DsRed. The difference in their molecular structures only wasapparent when the samples were boiled (denatured) before they wereloaded onto the gel. Boiled tDsRed migrated with an apparent molecularmass of about 65 kDa, which is approximately the mass of two DsRedmonomers, whereas boiled DsRed migrated at the monomer molecular mass of32 kDa.

A similar comparison was made for DsRed-I125R and tDsRed-I125R. Whenthey were not boiled prior to SDS-PAGE, tDsRed-I125R and DsRed-I125Rboth migrated as dimers with an apparent molecular mass of about 50 kDa.DsRed-I125R that was not boiled also had a large component that appearedto be denatured, though the fluorescent band for the dimer (50 kDa) wasclearly visible. tDsRed-I125R also had a denatured component thatmigrated slower (65 kDa vs. 50 kDa) than the intact fluorescent species.However, when boiled, tDsRed-I125R migrated at approximately the samemass as two monomers (65 kDa), while DsRed-I125R migrated at the monomermolecular mass of 32 kDa.

These results demonstrate that linking two DsRed monomers to form anintramolecularly bound tandem dimer prevented formation ofintermolecular oligomers, without affecting the emission or excitationspectra of the red fluorescent proteins.

Example 6 Red Fluorescent Proteins with Improvement Efficiency ofMaturation Wild-Type DsRed and Q66M DsRed Maturation

In an attempt to improve the speed and efficacy of maturation, Q66 ofdsRed was converted by site-directed mutagenesis to every othernaturally occurring amino acid. Only picking fluorescent colonies, itwas determined that almost all single-mutations at this position (F, N,G, T, H, E, K, D, R, L, Q were deleterious, leading to loss offluorescence, or inability to mature to a red fluorescent species. Onesubstitution, however, Q66M, yielded a protein that had significantlyimproved properties.

First, wild-type DsRed and Q66M DsRed were both produced in bacteria (E.coli) quickly purified, and allowed to mature in a cuvette sealed withparafilm. Parafilm was chosen to allow oxygen to diffuse into theprotein solution, and to prevent the loss of water from the solution.The maturation was monitored by taking absorbance scans over time. FIG.26 plots maturation as peak absorbance of the red chromophore versustime. Since the proteins were not expressed instantaneously or with theexact same efficiency, the two different curves did not both start atthe same value, and they reached slightly different end values. To allowcomparison, both curve amplitudes were first normalized to the samefinal absorbance. Then, the curves were fit to a three component kineticmodel, and the time base for each scan was adjusted so that the curvesintersected at zero time.

Wild-Type DsRed and Q66M DsRed Fluorescence

Fluorescence emission and excitation spectra of wild-type DsRed and theQ66M DsRed variant were taken using 558 nm excitation or monitoring 583nm emission for DsRed, and by using 566 nm excitation or monitoring 590nm emission for Q66M DsRed. The results are shown in FIG. 27. Of note isthe prominent red-shift of the entire Q66M DsRed fluorescence spectrum,as well as the marked depression of the excitation shoulder at 480 nmfor Q66M DsRed relative to wild-type.

Q66M DsRed Completeness of Maturation

Wild-type DsRed, Q66M DsRed, and another DsRed variant, K83R DsRed weresubjected to brief boiling in pH 1HCl, and then run on an SDSpolyactylamide gel. Since red chromophore formation makes the proteinacid labile at redicue 66, the relative amount of hydrolysis productsversus full-length protein is indicative of the completeness ofmaturation. The gel was Coomassie stained and imaged with a flat-bedscanner, and the relative intensity of all of the bands was thenquantified using the software NIH Image. After normalization formolecular weights (since the darkness of a Coomassie-stainer band isrelated to the mass of protein in the band, not the molarity), thematuration completeness was calculated by dividing the normalizedintensity of the split fragments' bands by the normalized intensity ofthe sum of all the bands. The results are shown in FIG. 28. As expectedfor K83R DsRed, which contains only a trace amount of red protein, onlyfull-length protein is visible. However, wild-type DsRed is degradedroughly ⅔ into split fragments, and Q66M DsRed is almost completelydegraded into split fragments.

In conclusion, when Q66M DsRed was expressed in bacteria, the bacteriaappeared to become fluorescent slightly faster than bacteria expressingwild-type DsRed. Furthermore, the Q66M DsRed protein appeared to bea-deeper pink color than wild-type DsRed, which has an orange tinge.Finally, the excitation spectrum of Q66M DsRed has a significantlysmaller hump at 480 nm than wild-type DsRed, suggesting that the speciesabsorbing at 480 nm (the immature green form of the protein) was is lessabundant. These data together show that Q66M DsRed has significantlyimproved properties over the wild-type DsRed protein.

Example 7 Preparation of a Further Improved Variant of the Monomeric RedFluorescent Protein, mRFP1

The engineering of the monomeric DsRed, mRFP 1 (SEQ ID NO: 8), asdescribed in the previous examples, has overcome at least three problemsassociated with the use of unmodified DsRed as a genetically encoded redfluorescent fusion label. Specifically, (1) mRFP1 is monomeric, (2) itmatures rapidly, and (3) is not efficiently excited at wavelengthssuitable for imaging of GFP. However mRFP1 is relatively dim (extinctioncoefficient (EC) of 44,000 M⁻¹ and quantum yield (QY) of 0.25) andtherefore limited in certain applications. In an effort to improve thebrightness of mRFP1 the strategy of directed evolution by randomizingspecific residues has been continued, in the hope of finding variantswith improved spectral properties.

In the first such library to improve the properties of mRFP 1 thefollowing codon substitutions were made in the mRFP1 cDNA:

N42Q to NNK=all 20 amino acids

V44A to NNK=all 20 amino acids

L46 to MTC=1 or L

Q66 to NNK=all 20 amino acids

K70 to ARG=K or R

V71A to GYC=V or A

This library contains a genetic diversity of 262,144 cDNAs which encodefor 64,000 different amino acid sequences. This library was transformedinto E. coli JM109(DE3) and bacterial colonies were manually screened(˜50,000 independent colonies) as described in the previous examples.Colonies that exhibited improved brightness or were a different colorwere picked and the gene was sequenced to determine the amino acidsubstitution. Top clones identified from this library fell into severaldifferent categories including:

Red-Shifted Variants:

-   -   Q66M+T147S; x588m610, EC ˜58,000 M⁻¹, QY ˜0.25    -   Q66M; x588m610, EC ˜52,000 M⁻¹, QY ˜0.25

Blue-Shifted Variants:

-   -   Q66T+Q213L; x574m595, EC ˜34,000 M⁻¹, QY ˜0.25    -   Q66T; x564m581, EC ˜23,000 M⁻¹, QY ˜0.25    -   Q66S; x558m578, dimmer than Q66T

Other Interesting Variants:

-   -   N42H+Q66G; x504m516, dim    -   V44M+Q66G; x504m516, dim    -   Q66L; absorbance maximum at 502 nm, practically non-fluorescent

The top mutant isolated from this library is mRFP1+Q66M/T147S, which hasbeen designated mRFP 1.1. Although this variant does not improve thequantum yield relative to mRFP1, mRFP1.1 is ˜30% brighter than mRFP1 dueto an improved EC. This improvement is due to an apparent increase inthe fraction of the protein that forms the mature red chromophore at theexpense of the non-fluorescent species that absorbs at 502 nm (see FIG.29, presenting the absorption and emission spectrum of mRFP1.1). Thebeneficial T147S mutant arose from an error during PCR amplification ofthe cDNA due to the use of Taq polymerise. The Q66M mutation haspreviously been shown to improve the fluorescence of the dimeric I125Rvariant of DsRed (Baird, G. S. (2001) Ph.D. Thesis, University ofCalifornia, San Diego). The amino acid sequence of mRFP1.1 is shown inFIG. 30 (SEQ ID NO: 79), while the nucleotide sequence of mRFP1.1 isprovided in FIG. 31 (SEQ ID NO: 80).

Example 8 Further Red Fluorescent Protein Variants

Further DsRed variants were produced using these methods. For example,FIG. 32 and TABLE 6 list some of these further variants. GFP termini areillustrated in FIG. 22B (SEQ ID NO: 14 represents the N-terminus and SEQID NO: 91 represents the C-terminus). An alternate variation of theC-terminal GFP sequence is provided in SEQ ID NO: 110.

TABLE 6 Designation Mutations Ex/Em EC QY Dimer2.2MMM(dimer3) Dimer2 +GFP termini + V22M, 554/581 70,000 0.72 (dTomato) Q66M, V104L, F124M(SEQ ID NO: 81) MRFP1.5 MRFP1.0 + GFP termini + 585/612 60,000 0.25 (SEQID NO: 83) ins(NNMA) after E6, VyI, T21S, Q66T, T147S, M182K, T195VOrS4-9 MRFP1.0 + GFP termini + 554/569 45,000 0.45 ± 0.05 (SEQ ID NO:85) ins(NNMA) after E6, V7I, T21S, Q66T, T147S, M182K, T195V Y1.3(mYOFP1.3) MRFP1.0 + GFP termini + 542/558 15,000 0.45 ± 0.05 (mBanana)ins(NNMA) after E6, Q66C, (SEQ ID NO: 87) A77T, D78G, L83F, T108A,T147S, D174S, V177T, M182K, K194I, T195A, D196G, I197E, L1991, Q213LmFRFP (F2Q6) MRFP1.0 + GFP termini + 605/632 40,000 0.03 mGrape2ins(NNMA) after E6, V7I, (SEQ ID NO: 89) E32K, Q66M, A77P, L83M, R125H,T147S, M150L, I161V, T195S, I197Y

The dimer variant dimer2.2MMM (dimer3) (dTomato) (SEQ ID NO:81) islisted in Table 6. It has the highest extinction coefficient, andimproved quantum yield compared to dimer2 (SEQ ID NO: 6). Dimer2.2MMM(dimer3) (dTomato) (SEQ ID NO:81) was also the dimer variant that hadthe extinction/emission ratio that was the most similar to the wild typeDsRed, and had less green component than either wild-type DsRed ordimer2 (SEQ ID NO: 6). However, it does still have a residual greencomponent.

Several monomer variants are also listed in Table 6. The monomer variantmRFP1.5 (SEQ ID NO: 83), had an emission peak that was red-shifted byabout 5 nm, and significantly reduced green component, as compared withmRFP1.0. It also showed nearly complete maturation. However, its quantumyield is not extremely high. The monomer variant OrS4-9 (SEQ ID NO:85)was blue-shifted relative to mRFP and to wild-type. DsRed, with a higherquantum yield than mRFP1.0 or mRFP1.5, and had reduced pH sensitivity ascompared with the original orange mRFP variants. However, its extinctioncoefficient is low, possibly due to difficulty in folding or incompletematuration. The monomer variant Y1.3 (mYOFP1.3) (mBanana) (SEQ ID NO:87)had the largest blue-shift of any mRFP variant. It also had the highestquantum yield and a reduced pH sensitivity as compared with the originalyellow mRFP variants, although its extinction coefficient is also low,possibly due to incomplete maturation. The monomer variant mFRFP (F2Q6)(mGrape2) (SEQ ID NO: 89) had the largest red-shift of any mRFP variant,and seems to have nearly complete maturation, although its quantum yieldand its extinction coefficient are not extremely high.

Other monomeric variants may also be useful. For example, in someembodiments, a useful protein variant is selected from the group ofvariants including mRFP1.5 (SEQ ID NO: 83), OrS4-9 (SEQ ID NO: 85), Y1.3(mYOFP1.3) (mBanana) (SEQ ID NO: 87), mFRFP (F2Q6) (mGrape2) (SEQ ID NO:89), mRFP2 (mCherry) (SEQ ID NO: 92), mOFP (74-11) (SEQ ID NO: 94),mROFP (A2/6-6) (SEQ ID NO: 96), mStrawberry (SEQ ID NO: 98), mTangerine(SEQ ID NO: 100), mOrange (mOFP1) (SEQ ID NO: 102), mHoneydew (SEQ IDNO: 104), and mGrape1 (SEQ ID NO: 108).

As indicated above, dimeric variants may also be useful, and may includedimers including one or more of the monomers indicated above. Dimericvariants may also include a dimer subunit that is selected from, forexample, Dimer1, dimer1.02, Dimer1.25, .dimer1.26, dimer 1.28,Dimer1.34, Dimer1.56, Dimer1.61, Dimer1.76, dimer2, dimer 2.2MMM (dimer3) (dTomato) SEQ ID NO: 81) and tdTomato (SEQ ID NO: 106).

Example 9 Further Red Fluorescent Protein Variants

This example provides further discussion relating to the monomervariants. For example, one of the latest red versions matures morecompletely, is more tolerant of N-terminal fusions, and is over tenfoldmore photostable than mRFP1. Three new monomers with distinguishablehues from yellow-orange to red-orange have higher quantum efficienciesand suitability as re-emitting energy transfer acceptors.

Although mRFP 1 overcame DsRed's tetramerization and sluggish maturationand exceeded DsRed's excitation and emission wavelengths by about 25 nm,the extinction coefficient, fluorescence quantum yield, andphotostability decreased somewhat during the evolution of mRFP1⁷. Tominimize these sacrifices, we have now subjected mRFP1 to many rounds ofdirected evolution using both manual and FACS-based screening. Theresulting plethora of new variants include several new colors, increasedtolerance of N- and C-terminal fusions, and improvements in extinctioncoefficients, quantum yields, and photostability, though no singlevariant is optimal by all criteria.

mCherry. The red chromophore of DsRed results from the autonomousmulti-step post-translational modification of residues Gln66, Tyr67, andGly68 into an imidazolidinone heterocycle with p-hydroxybenzylidene andacylimine substituents⁸. Based on the fact that introduction of Met atposition 66 had a beneficial effect on the degree of maturation of theI125R dimeric variant of DsRed⁹, our first attempt at improving thebrightness of mRFP1 involved construction of a directed library in whichresidues near the chromophore, including position 66, were randomized.The top clone of this library, mRFP 1.1, contains the mutation Q66M(along with the complementary T147S), which, in addition to promotingmore complete maturation, provides an additional 5 nm red-shift of boththe excitation and emission spectra relative to mRFP1. We then noticedthat the red fluorescence of both mRFP1 and mRFP1.1 could be drasticallyreduced by the presence of certain N-terminal fusions. Similarly, otherresearchers had noted that point mutations in the first few residues oftetrameric DsRed could provide dramatic increases in the observedfluorescent intensity¹⁰. We reasoned that since the N-terminal sequenceof mRFP1 had been originally optimized for solubility of the unfusedprotein⁶ rather than ability to tolerate fusions, this relativelyarbitrary sequence could be far from ideal. Because Aequorea GFP isrelatively indifferent to N- or C-terminal fusions, we eventuallygenerated mRFP1.3 by replacing the first six amino acids of mRFP 1.1with the corresponding residues from enhanced GFP (MVSKGEE, (SEQ IDNO:14)) followed by a spacer sequence NNMA (SEQ ID NO:112), andappending the last seven amino acids of GFP to the C-terminus. The 4inserted residues NNMA (SEQ ID NO:112) are numbered 6a-d to preserveDsRed numbering for the rest of the protein.

Additional rounds of screening random libraries based on mRFP1.3 andwavelength-shifted mRFP variants identified the beneficial foldingmutations V7I and M182K, which were incorporated into clone mRFP1.4. Alibrary randomized at position 163 identified the substitution M163Q,resulting in a nearly complete disappearance of the absorbance peak at˜510 nm present in all previous mRFP clones, but a reduction in foldingefficiency. The additional mutations R17H and N6aD (a substitution of Nat position 6a, the numbering as explained in paragraph [0219], with D)restored an extinction coefficient to nearly equivalent to mRFP1.4. Thisvariant, designated mRFP1.5, had excitation and emission 3 nmred-shifted from mRFP1 and exhibited nearly complete maturation withkinetics as fast or faster than mRFP1. Finally, an additional directedlibrary constructed by partially randomizing positions 194, 195, 196,197, and 199, resulted in the identification of a clone containing themutations K194N, T195V, and D196N, which was found to have an enhancedextinction coefficient, while retaining the nearly complete lack of a510 nm absorbance peak. This final clone was designated mCherry (SeeTable 7, FIGS. 47A and B, FIGS. 48A and B, and FIG. 50).

dTomato and tdTomato. The dimer2 variant previously described⁷ possessesmany desirable properties such as a faster and more complete maturationthan wild-type DsRed and a greater fluorescent brightness than thefast-maturing mutant T1⁶ (DsRed-Express). Through 5 rounds of directedevolution, we found the optimal combination of mutations V22M, Q66M,V105L, and F124M, which resulted in improved maturation kinetics, asignificantly reduced ‘dead-end’ green component, and a small red-shift.The final clone, designated dTomato (see Table 7 and FIGS. 47A and 47B),additionally contains the GFP-type termini as described for mCherry(without the NNMA insertion), which result in a higher tolerance of N-and C-terminal fusions (data not shown). In order to construct anonaggregating tag from the extremely bright dTomato, we geneticallyfused two copies of the gene to create a tandem dimer as previouslyreported. GFP-type termini were included at the N-terminus of the firstcopy of dTomato and at the C-terminus of the second copy of dTomato. TheN-terminus of the second copy of dTomato consists of amino acids 2through 6 of dimer2 followed by the NNMA insertion. This arrangement,designated tdTomato, provided the highest level of expression in E. coliwhile maintaining all of the desirable properties of the dimericdTomato.

Wavelength-shifted variants of mRFP. Many new FPs with different colorshave been discovered in diverse anthozoan species, but so far they allsuffer from obligate tetramerization and would require efforts similarto the evolution of mRFP1 to produce widely useful fusion partners¹¹Because such monomerization is usually tedious and often unsuccessful,it might be more efficient to alter the excitation and emissionwavelength of mRFP through directed evolution. Following the example ofGFP engineering^(12, 13), we explored substitutions at Tyr 67,homologous to Tyr 66 in GFP, which contribute the core of thechromophore, and at Gln66, homologous to Ser 65 of GFP, which are thenext most important contributors.

Starting with mRFP1.1, we replaced Tyr67 with either Phe, His, or Trp.The most promising clone contained the Tyr67Trp substitution, homologousto the main wavelength-shifting mutation in the cyan GFP mutant, CFP.Two further rounds of directed mutagenesis yielded our most blue-shiftedmRFP variant, named “mHoneydew” which contains four additionalsubstitutions (see FIG. 48A and FIG. 48B), but does not have optimizedN- and C-termini. Like CFP, mHoneydew has relatively broad,double-peaked excitation and emission spectra, with excitation peaks at487 and 504 nm, and emission peaks at 537 and 562 nm. Its extinctioncoefficient (17,000 M⁻¹cm⁻¹) and quantum yield (0.12) are quite low andproved relatively resistant to further improvements in initiallibraries, and so was not evolved further in this study. Nevertheless,mHoneydew proves that the tryptophan-based chromophore of CFP canundergo a further maturation to a longer-wavelength chromophore, inanalogy to the dehydrogenation of the tyrosine-derived GFP chromophoreto the DsRed/mRFP chromophore.

Initial mutations of position 66 in mRFP1.1 indicated that thesubstitutions Gln66Ser, Gln66Thr, and Gln66Cys all yielded proteinssignificantly blue-shifted with respect to mRFP1. This led us to thedevelopment of “mTangerine,” which contains the substitutions Gln66Cysand Gln213Leu with respect to mRFP1.1 (see FIGS. 48A and B), withexcitation and emission peaks at 568 and 585 nm. While mTangerine has arespectable extinction coefficient (38,000 M⁻¹cm⁻¹) and quantum yield(0.3), we quickly moved on to development of Gln66-substituted mutantsof mRFP1.4, with its optimized N- and C-termini.

In mRFP1.4, the substitutions Gln66Ser, Gln66Thr, and Gln66Cys all gavea similar blue-shift in both excitation and emission wavelength, and ledto a curious increase in pH sensitivity that was characterized by anadditional blue-shift at alkaline pH. Starting with mRFP1.4 M66T,through 6 additional rounds of directed, evolution, we arrived at thefinal orange fluorescent variant, mOrange, with excitation at 548 nm andemission at 562 nm (see Table 7, FIGS. 47A, 47B, 48A and 48B). mOrangehas an extinction coefficient equivalent to mCherry, but more than3-fold higher quantum yield. Interestingly, mOrange has excitation andemission maxima very close to a newly discovered tetrameric orange FPfrom Cerianthus ¹⁴ and a monomer evolved from a Fulgia concinna FP¹⁵.Though mOrange is the brightest true monomer in the present series, itdoes exhibit significant acid sensitivity, with a pKa of 6.5, and so isnot yet optimal when pH insensitivity is required. However, the popularAequorea GFP variant EYFP, with a pKa of 7.1¹⁶, has been usedsuccessfully as a qualitative fusion tag by many researchers.Additionally, from the initial mRFP1.4 M66T clone, through 5 rounds ofdirected evolution, we created a pH-stable orange-red variant in whichthe longer-wavelength form of the Q66T chromophore has been enhanced.This variant, mStrawberry, has the highest extinction coefficient(90,000 M⁻¹cm⁻¹) of the true monomers. Its wavelengths (excitation 574nm, emission 596 nm) and quantum yield (0.29) are intermediate betweenmCherry and mOrange (see Table 7, FIGS. 47A and B, and FIGS. 48A and B).

The high extinction coefficient and quantum yield of mOrange made itattractive as a potential FRET acceptor for GFP variants. As a test, we(assisted by Amy Palmer) constructed a new Zn²⁺ sensor with mOrange andthe violet-excited GFP mutant T-Sapphire¹⁷ and compared it to the samesensor containing CFP and the Citrine variety¹⁸ of YFP. T-Sapphire waschosen as donor because it is optimally excited below 425 nm, wheremOrange is negligibly excited. The domain that changes its conformationupon binding Zn²⁺ was modified from the original zif-268-derivedversion¹⁹ by mutating the two Zn²⁺-binding Cys to His to eliminate anypossibility of oxidation. The fusion of CFP and Citrine respectively tothe N- and C-terminii of the modified Zn²⁺ finger displayed a 5.2 foldratio change upon addition of Zn²⁺, with an apparent K_(d) of ˜200 μM.Replacement of CFP by mOrange and Citrine by T-Sapphire yielded a sensorwhose 562 nm to 514 nm emission ratio increased 6-fold upon Zn²⁺ binding(FIG. 49). This demonstrates that mOrange and T-Sapphire make a goodemission-ratiometric FRET pair with dynamic range at least equaling CFPand YFP but at longer emission wavelengths.

Position 197 stacks near the RFP chromophore in homology to Thr203 inGFP. Randomization of Ile 197 in mTangerine led to the surprisingdiscovery that Glu provided an additional blue-shift. This variant wasinitially found to have extreme pH sensitivity as well as inefficientmaturation. Eight rounds of directed evolution gradually improved thefolding efficiency and pH sensitivity, eventually yielding mBanana, withexcitation and emission peaks at 540 nm and 553 nm respectively and ahigh quantum yield (0.70). Unfortunately the fluorescence of mBanana isquite pH-sensitive, and the effective extinction coefficient remainsquite low, only 6,000 M⁻¹cm⁻¹, probably due to incomplete maturation.Most of the protein seems trapped as a dead-end species absorbing at 385nm. However, the increased quantum yield of mBanana partiallycompensates for its low extinction coefficient (see Table 7, FIGS. 47Aand B, and FIGS. 48A and B).

What is the basis of the spectral shifts of these mRFP variants? Thelargest single effect is seen with substitutions at the firstchromophore position, occupied by Gln in DsRed and mRFP1. Substitutionof this residue with Ser, Thr, or Cys in mRFP1 or dimeric RFPs (data notshown) results in a significant blue-shift. In the absence ofcompensating mutations, the Q66S/T/C variants of mRFP1 all exhibitemission around 580 nm at neutral pH, but become brighter and moreblue-shifted at high pH. The blue-shifted (mOrange) species isstabilized by T41F and L83F mutations, where the red-shifted(mStrawberry) form is favored by S62T, Q64N, and Q213L. In mBanana, theI197E mutation may contribute a hydrogen bond between the glutamate sidechain and phenolate oxygen in the chromophore, resulting in a furtherredistribution of electron density.

Discrimination of Six FPs

An obvious application of the proliferation of FP colors is todiscriminate many cell types, transcriptional activities, or fusionproteins. In the most general case where a cell or voxel could containarbitrary concentrations of n FPs mixed together, determination of the nindependent concentrations or quantities of each of the FPs requiresspectral or lifetime unmixing of at least n measurements, preferablymany more to confer statistical robustness. However, there isan-important special case where each cell or nonoverlapping subcellularstructure has been tagged with a different FP. When a cell or voxelcontains at most one FP, how many measurements are required to decideits identity and concentration? To test such discriminationexperimentally, bacteria separately transfected with EGFP, Citrine,mBanana, mOrange, mStrawberry, or mCherry were mixed and analyzed byflow cytometry, with excitation at 514 nm and emission simultaneouslymeasured through three bandpass filters, 540-560 nm, 564-606 nm, and595-635 nm respectively. Transformation of the three signals to polarcoordinates enabled easy discrimination of the six constituentpopulations and the relative amounts of FP per cell (FIG. 52). Thusthree simple emission measurements are sufficient.

Photostability Measurements

We compared the photostability of mRFP1 with that of its descendants andwith EGFP by making microdroplets of purified protein solutions inphosphate-buffered saline under mineral oil, illuminating them on afluorescence microscope at irradiances of several watts/cm², andrecording the time course of gradual loss of fluorescence due tobleaching (see Methods below). In order to make fair comparisons betweenproteins with different extinction coefficients, overlaps withexcitation filters, and quantum yields, we normalized the bleach ratesto an initial emission rate from each molecule of 1000 photons/sec. Theresulting bleach curves (shown in FIG. 51) varied greatly in absoluterate but were generally far from single exponentials. Most showed aninitial fast phase in which over 50% of the initial emission was lost,followed by a lower amplitude, much slower decay phase. This complexitymakes it difficult to extract a meaningful single number for the quantumefficiency of photobleaching. Another common previous figure of meritfor fluorophore photostability is the mean number of photons emittedbefore photobleaching, ideally corresponding to the total area undereach curve. However, this integral is usually dominated by the dimslow-bleaching component, whose low intensity would hinderdiscrimination from background autofluorescence in a real biologicalexperiment, and which might not even have the same excitation andemission spectra as the original protein. We believe a more realisticfigure of merit for typical cell biological experiments is the time forthe emission to drop to 50% of its initial value. These values arelisted in Table 7. By this criterion, the best performers are tdTomatoand mCherry, which are both more than tenfold better than mRFP1 andnearly as good as EGFP.

For example, we investigated the mutations responsible for increasedphotostability of mCherry relative to mRFP1 by measuring thephotostability of several intermediate mutants. We found that theincrease in photostability occurred between the mutants mRFP1.4 andmRFP1.5, which differ by the N6aD (a substitution of N at position 6a,the numbering as explained in paragraph [0219], with D), R17H, and M163Qmutations, of which only M163Q is internal and interacts with thechromophore, and so is likely to be responsible for the increase inphotostability. The photostability of mRFP1.5 and mCherry is practicallyidentical, indicating that the mutations that occurred between these twovariants had no effect on the photostability. When the mutation M163Q isintroduced into other variants, such as mOrange and mStrawberry, it alsoprovides a significant increase in photostability, though otherproperties of these variants, such as the maturation rate and quantumyield, are adversely affected by this mutation.

The highest brightness (product of extinction coefficient, 138,000M⁻¹cm⁻¹, and quantum yield, 0.69) and photostability are found intdTomato, at the cost of doubling the molecular weight. A previoustandem dimer, t-HcRed1, was reported to have an extinction coefficientand quantum yield of 160,000 M⁻¹cm⁻¹ and 0.04²⁰, whose product is about15-fold smaller than tdTomato's. Among the true monomers, mCherry offersthe longest wavelengths, the highest photostability, the fastestmaturation, and excellent pH resistance. Its excitation and emissionmaxima are just 3 nm longer than those of mRFP1, for which it is theclosest upgrade. Although mCherry's quantum efficiency is slightly lower(0.22 vs. 0.25 for mRFP1), its increased extinction coefficient (due tonear-complete maturation), tolerance of N-terminal fusions, andphotostability make mRFP1 obsolete. For applications such asdual-emission FRET where the acceptor's quantum yield must be maximized,mOrange is the current favorite (e.g. FIG. 49), though its maturationtime, pH sensitivity, and photostability are currently far from optimal.Additional colors for multiwavelength tracking of distinct cells orsubstructures are available from mStrawberry, mTangerine, mBanana, andmHoneydew in descending order of wavelengths and brightnesses.

Methods

Mutagenesis and Screening. mRFP1 and dimer2⁷ were used as the initialtemplates for construction of genetic libraries by a combination ofsaturation or partial saturation mutagenesis at particular residues andrandom mutagenesis of the whole gene. Random mutagenesis was performedby error-prone PCR as described¹⁸ or by using the GeneMorph I orGeneMorph II kit (Stratagene). Mutations at specific residues wereintroduced as described, or by sequential QuikChange (Stratagene), or byQuikChange Multi (Stratagene), or by a ligation-based method(description follows). Briefly, oligonucleotide primers containing thedegenerate codons of interest at their 5′ ends preceded by a SapIrestriction site were used to amplify the RFP in two separate PCRreactions using PfuTurbo polymerase (Stratagene). Each PCR fragment wascut with SapI (New England Biolabs) to produce a 3-base overhangcompatible with the other digested fragment, and purified digestedfragments were ligated with T4 DNA ligase (New England Biolabs). Fulllength ligation products inserts were gel purified and cut withEcoRI/BamHI (New England Biolabs) and inserted into pRSET_(B) or amodified pBAD vector (Invitrogen). For all library construction methods,chemically competent or electrocompetent Escherichia coli strainJM109(DE3) (for pRSET_(B)) or LMG194 (for pBAD)²⁶ were transformed andgrown overnight on LB/agar (supplemented with 0.02% L-arabinose (Fluka)for pBAD constructs) at 37° C. and maintained thereafter at roomtemperature. LB/agar plates were manually screened as previouslydescribed²⁷. JM109(DE3) colonies of interest were cultured overnight in2 ml LB supplemented with ampicillin. LMG194 colonies of interest werecultured for 8 hours in 2 ml RM supplemented with ampicillin and 0.2%D-glucose, and then culture volume was increased to 4 ml withLB/ampicillin and RFP expression was induced by adding L-arabinose to afinal concentration of 0.2% and cultures were allowed to continuegrowing overnight. For both JM109(DE3) and LMG194, a fraction of thecell pellet was extracted with B-PER II (Pierce), and spectra wereobtained using a Safire 96-well plate reader with monochromators (TECAN,Männendorf, Switzerland). DNA was purified from the remaining pellet byQIAprep spin column (Qiagen) and submitted for sequencing.

Construction of Tandem Dimer and FRET Constructs. To construct tdTomatowith a 12-residue linker (GHGTGSTGSGSS), dRFP3 was amplified in twoseparate PCR reactions, the first retaining the 7-residue GFP-typeN-terminus (MVSKGEE) but deleting the 7-residue GFP-type C-terminus andadding the first half of the 12-residue linker followed by a SapIrestriction site, and the second adding the remaining half of the12-residue linker followed by the sequence ASSEDNNMA before residue 7 ofdRFP3, and ending with the 7-residue GFP-type C-terminus (GMDELYK). Gelpurified PCR products were digested with SapI, gel purified, and ligatedwith T4 DNA ligase. Full length ligation product was gel purified, cutwith EcoRI/BamHI, and ligated into a modified pBAD vector. For FRETconstructs, the original Zn²⁺ sensor¹⁹ was modified such that the twoZn²⁺ ligating Cys residues were mutated to His. The modified Zn²⁺ fingerdomain, HERPYAHPVESHDRFSRSDELTRHIRIHTGQK (Zn²⁺ ligating residues inbold, mutated residues underlined), was cloned between CFP and citrinewith SphI and SacI sites as linkers. PCR-amplified mOrange andT-Sapphire¹⁷ were inserted into BamHI/SphI and EcoRI/SacI sites,replacing CFP and citrine.

Protein Production and Characterization. RFPs were expressed from pBADvectors in E. coli LMG194 by growing single colonies in 40 ml RM/Ampsupplemented with 0.2% D-glucose for 8 hours, adding 40 ml LB/Amp andadding L-arabinose to a final concentration of 0.2%, and incubatingovernight at 37° C. For maturation experiments, flasks were sealed withparafilm upon induction to restrict oxygen availability. All proteinswere purified by Ni-NTA chromatography (Qiagen) and dialyzed into PBS.Biochemical and fluorescence characterization experiments were performedas described. For FRET measurements, purified Zn²⁺ sensor proteins werediluted in 10 mM MOPS, 100 mM KCl, pH 7.4 with either 1 mM EDTA or 1 mMZnCl₂ and fluorescence emission spectra were collected with excitationnear the peak donor excitation wavelength.

Fluorescence Activated Cell Sorting. A modified version of the protocoldescribed by Daugherty et al. was used for FACS screening of largelibraries of FP mutants²⁸. Briefly, E. coli LMG194 were electroporatedwith a modified pBAD vector containing the gene library and thetransformed cells were grown in 30 ml RM supplemented with ampicillinand 0.2% D-glucose. After 8 hours, RFP expression was induced by addingL-arabinose to a final concentration of 0.2%. Overnight induced cultureswere diluted 1:100 into DPBS supplemented with ampicillin prior to FACSsorting. Multiple rounds of cell sorting were performed on a FACSDiva(BD Biosciences) in yield mode for the first sort and purity or singlecell mode for subsequent sorts of the same library. Sorted cells weregrown overnight in 4 ml RM/amp with 0.2% D-glucose and the resultingsaturated culture was diluted 1:100 into 30 ml RM/Amp with 0.2%D-glucose to start the next culture to be sorted. After 3 to 4 rounds ofFACS sorting, the bacteria were plated onto LB/agar supplemented with0.02% L-arabinose and grown overnight, after which individual cloneswere screened manually as described above.

Photobleaching measurements. Aqueous droplets of purified protein inphosphate-buffered saline were formed under mineral oil in a chamber onthe fluorescence microscope stage. For reproducible results it provedessential to pre-extract the oil with aqueous buffer, which would removeany traces of autoxidized or acidic contaminants. The droplets weresmall enough (5-10 μm diameter) so that all the molecules would see thesame incident intensity. The absolute excitation irradiance inphotons/(cm²·s·nm) as a function of wavelength was computed from thespectra of a xenon lamp, the transmission of the excitation filter, thereflectance of the dichroic mirror, the manufacturer-supplied absolutespectral sensitivity of a miniature integrating-sphere detector (SPD024head and ILC1700 meter, International Light Corp., Newburyport, Mass.),and the measured detector current. The predicted rate of initial photonemission (before any photobleaching had occurred) was calculated fromthe excitation irradiance and absorbance spectrum (both as functions ofwavelength), and the quantum yield. These rates varied from 180 s⁻¹ formHoneydew to 3300 s⁻¹ for mStrawberry. To normalize the observedphotobleaching time courses to a common arbitrary standard of 1000emitted photons/sec, the time axes were correspondingly scaled byfactors of 0.18 to 3.3, assuming that emission and photobleach rates areboth proportional to excitation intensity at intensities typical ofmicroscopes with arc lamp sources, as is known to be the case for GFP²⁹.

TABLE 7 Brightness Extinction of fully Excitation Emission coefficientFluorescence mature t_(0.5) for t_(0.5) for Fluorescent maximum,maximum, per chain^(a), quantum protein (% maturation bleach^(b),protein nm nm M⁻¹cm⁻¹ yield of DsRed) pK_(a) at 37° C. sec DsRed 558 58375,000 0.79 100 4.7 ~10 hr ND T1 555 584 38,000 0.51 33 4.8  <1 hr NDDimer2 552 579 69,000 0.69 80 4.9  ~2 hr ND mRFP1 584 607 50,000 0.25 214.5  <1 hr 6.2 mHoneydew 487/504 537/562 17,000 0.12 3 <4.0 ND 5.9mBanana 540 553 6,000 0.70 7 6.7  1 hr 1.4 mOrange 548 562 71,000 0.6983 6.5  2.5 hr 6.4 dTomato 554 581 69,000 0.69 80 4.7  1 hr, 64.tdTomato 554 581 138,000 0.69 160 4.7  1 hr 70. mTangerine 568 58538,000 030 19 5.7 ND 5.1 mStrawberry 574 596 90,000 0.29 44 <4.5  50 min11. mCherry 587 610 72,000 0.22 27 <4.5  15 min 68. ^(a)Extinctioncoefficients were measured by the alkali denaturation method and arebelieved to be more accurate than the previously reported values forDsRed, T1, dimer2, and mRFP1⁷ ^(b)Time (s) to bleach to 50% emissionintensity, at an illumination level that causes each molecule to emit1000 photons/sec initially, i.e. before any bleaching has occurred. SeeMaterials and Methods for more details. For comparison, the value forEGFP is 115 sec, assuming an extinction coefficient of 56,000 andquantum efficiency of 0.60.

Figure Legends Referred to in Example 9

FIGS. 47A and 47B: Excitation and emission spectra for new RFP variants.Spectra are normalized to the excitation and emission peak for eachprotein. Excitation (a) and emission (b) curves are shown as solid ordashed lines for mRFP1 variants and as a dotted line for dTomato andtdTomato, with colors corresponding to the color of each variant.

FIGS. 48A and 48B: Sequences and genealogy. (a) Sequence alignment ofnew mRFP variants with wild-type DsRed and mRFP1. Internal residues areshaded. mRFP1 mutations are shown in blue, and critical mutations inmCherry, mStrawberry, mTangerine, mOrange, mBanana, and mHoneydew areshown in colors corresponding to the color of each variant. GFP-typetermini on new mRFP variants are shown in green. (b) A genealogy ofDsRed-derived variants, with mutations critical to the phenotype of eachnew variant.

FIG. 49: T-Sapphire-mOrange FRET. Emission spectra for 400 nm excitationfor a zinc-finger fused with mOrange on its N-terminus and T-Sapphire onits C-terminus. Emission in the presence of 1 mM EDTA in zinc-freebuffer is represented by the green line, and emission in the presence of1 mM ZnCl₂ is represented by the orange line.

FIG. 50: Sensitivity to N- and C-terminal fusions. mRFP1 and mCherryC-/N-terminal 6×His tag absorbance spectra. mRFP 1 and mCherry withN-terminal leader sequence containing a 6×His tag (derived frompRSET_(B)) or C-terminal tail with Myc-tag and 6×His tag (derived frompBAD-Myc-His-A (Invitrogen)) were purified on Ni-NTA Agarose beads inparallel with extensive washes. Absorbance spectra were taken andnormalized to the 280 nm peak for each. Absorbance curves for mRFP1 areplotted in red, and those from mCherry are plotted in blue. Solid linescorrespond to N-terminal 6×His-tagged protein, and dotted linescorrespond to C-terminal 6×His-tagged protein. While mRFP 1 exhibitedgreatly reduced expression and apparent extinction coefficient(approximately 4-fold lower) when expressed with the C-terminal tag,mCherry produced nearly identical results with either N-terminal orC-terminal tags.

FIG. 51: Photobleaching curves for new RFP variants. Curves for mRFP1and EGFP are included for comparison. All curves are normalized toillumination intensities calculated to cause each molecule to emit 1000photons/sec at time=0 before photobleaching.

FIG. 52: Discrimination of E. coli transfected with six different FPs.Bacteria separately transformed with different FPs were mixed andanalyzed by flow cytometry for a total of about 10,000 events.Excitation was at 514 nm from an argon-ion laser. Emissions E₁, E₂, andE₃ were simultaneously collected with 540-560 nm, 564-606 nm, and595-635 nm bandpass filters respectively. Normalized fluorescences F_(i)were calculated as E_(i)/(mean of all E_(i) values) for i=1, 2, 3. Theroot-mean-square intensity (ordinate) was defined as [E₁ ²+E₂ ²+E₃²]^(1/2), while the angle sum (abscissa) was tan⁻¹ (E₂/E₁)+tan⁻¹ (E₃/E₂)in radians. Each dot represents a cell. Control runs with purepopulations verified that the blue-green dots represent GFP-expressingcells, green dots mean Citrine YFP, yellows mean mBanana, oranges meanmOrange, reds mean mStrawberry, and magentas mean mCherry.

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All publications, GenBank Accession Number sequence submissions, patentsand published patent applications mentioned in the above specificationare herein incorporated by reference in their entirety. Variousmodifications and variations of the described compositions and methodsof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with various specificembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in protein chemistry ormolecular biological arts or related fields are intended to be withinthe scope of the following claims.

1. An isolated polynucleotide encoding a Discosoma red fluorescentprotein (DsRed) variant, wherein the variant comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:8.
 2. Thepolynucleotide of claim 1, wherein the variant comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:8.
 3. Thepolynucleotide of claim 1, wherein the encoded DsRed variant hasimproved fluorescence maturation relative to the polypeptide of SEQ IDNO:1.
 4. The polynucleotide of claim 1, wherein the encoded DsRedvariant is a monomer.
 5. A vector comprising a polynucleotide ofclaim
 1. 6. The vector of claim 5, wherein the vector is an expressionvector.
 7. A host cell comprising the vector of claim
 5. 8. A kit,comprising a polynucleotide of claim 1.